LOCUS       Cas9-T6N      11171 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of cell permeable spCas9 in E. Coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11171)
  AUTHORS   Zhang Z, Baxter AE, Ren D, Qin K, Chen Z, Collins SM, Huang H, Komar
            CA, Bailer PF, Parker JB, Blobel GA, Kohli RM, Wherry EJ, Berger SL,
            Shi J
  TITLE     Efficient engineering of human and mouse primary cells using 
            peptide-assisted genome editing.
  JOURNAL   Nat Biotechnol. 2023 Apr 24. doi: 10.1038/s41587-023-01756-1.
  PUBMED    37095348
REFERENCE   2  (bases 1 to 11171)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2023 Apr 24. doi: 10.1038/s41587-023-01756-1."
COMMENT     Sequence Label: Cas9-T6N (pZZ12, TAT-4MycNLS-Cas9-2SV40NLS-sfGFP)
FEATURES             Location/Qualifiers
     source          1..11171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1328..2410)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2372..2391
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2411..2488)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2694..2713
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2797..2816
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2797..2815
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2816..2840
                     /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)."
     RBS             2855..2877
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2896..2913
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2923..2940
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(2953..2976,2977..3012,3013..3036)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             3037..3330
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3379..3405
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             3415..3441
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             3451..3477
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             3487..3513
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             3541..7644
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3541..7644
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7651..7671
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7678..7698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(8468..8486)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8482..8529
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8704..8709,8710..8726,8727..8732))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(8759..8777)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8845..8949
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8950..9018,9019..9810)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9168..9187)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9981..10569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10470..10489
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10723..10740
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    10755..10894
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(10820..10839)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10980..11002
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(10996..11171,1..16))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
       61 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
      121 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
      181 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
      241 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
      301 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
      361 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
      421 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
      481 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
      541 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
      601 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
      661 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
      721 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
      781 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
      841 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
      901 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
      961 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
     1021 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     1081 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     1141 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     1201 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     1261 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     1321 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1381 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     1441 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     1501 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     1561 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     1621 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     1681 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     1741 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     1801 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     1861 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     1921 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     1981 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     2041 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     2101 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     2161 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     2221 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     2281 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     2341 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     2401 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     2461 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     2521 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     2581 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     2641 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     2701 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     2761 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     2821 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     2881 accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc
     2941 catatggcta gctggagcca tccgcagttt gaaaaaggtg gtggtagcgg tggtggttca
     3001 ggtggtagtg catggtcaca ccctcagttt gagaaaatgt cggactcaga agtcaatcaa
     3061 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
     3121 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
     3181 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
     3241 ggtattagaa tccaagctga tcagacccct gaagatttgg acatggagga taacgatatt
     3301 attgaggctc acagagaaca gattggtgga tccggccgta agaaacgccg ccagcgtcgc
     3361 cgtccgcaag gcggtagccc cgccgcgaag cgtgtaaagc tggatggggg tagcccagcg
     3421 gcaaaaaggg tgaaactaga cggcggctcc ccggcagcca agagagtcaa actcgacggg
     3481 ggtagccctg ctgctaaacg tgttaagctt gatggtatcc acggagtccc agcagctacc
     3541 atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg
     3601 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc
     3661 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa
     3721 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt
     3781 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga
     3841 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga
     3901 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa
     3961 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat
     4021 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat
     4081 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct
     4141 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga
     4201 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat
     4261 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa
     4321 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg
     4381 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt
     4441 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca
     4501 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga
     4561 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca
     4621 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta
     4681 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc
     4741 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat
     4801 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt
     4861 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt
     4921 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa
     4981 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa
     5041 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt
     5101 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt
     5161 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc
     5221 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt
     5281 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt
     5341 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt
     5401 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct
     5461 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga
     5521 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta
     5581 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat
     5641 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta
     5701 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact
     5761 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt
     5821 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt
     5881 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct
     5941 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga
     6001 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac
     6061 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct
     6121 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa
     6181 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta
     6241 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa
     6301 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat
     6361 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct
     6421 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat
     6481 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa
     6541 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa
     6601 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct
     6661 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc
     6721 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt
     6781 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta
     6841 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt
     6901 gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct
     6961 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt
     7021 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac
     7081 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa
     7141 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta
     7201 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt
     7261 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag
     7321 cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt
     7381 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa
     7441 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct
     7501 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa
     7561 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt
     7621 gatttgagtc agctaggagg tgacgggtca cctaagaaaa aacgaaaagt tgaggatcct
     7681 aaaaagaaac gaaaagttga taccggttct aaaggtgaag aactgttcac cggtgttgtt
     7741 ccgatcctgg ttgaactgga tggtgatgtt aacggccaca aattctctgt tcgtggtgaa
     7801 ggtgaaggtg atgcaaccaa cggtaaactg accctgaaat tcatctgcac taccggtaaa
     7861 ctgccggttc catggccgac tctggtgact accctgacct atggtgttca gtgtttttct
     7921 cgttacccgg atcacatgaa gcagcatgat ttcttcaaat ctgcaatgcc ggaaggttat
     7981 gtacaggagc gcaccatttc tttcaaagac gatggcacct acaaaacccg tgcagaggtt
     8041 aaatttgaag gtgatactct ggtgaaccgt attgaactga aaggcattga tttcaaagag
     8101 gacggcaaca tcctgggcca caaactggaa tataacttca actcccataa cgtttacatc
     8161 accgcagaca aacagaagaa cggtatcaaa gctaacttca aaattcgcca taacgttgaa
     8221 gacggtagcg tacagctggc ggaccactac cagcagaaca ctccgatcgg tgatggtccg
     8281 gttctgctgc cggataacca ctacctgtcc acccagtctg ttctgtccaa agacccgaac
     8341 gaaaagcgcg accacatggt gctgctggag ttcgttactg cagcaggtat cacgcacggc
     8401 atggatgagc tctacaaata agcggccgca ctcgaggccc gaaaggaagc tgagttggct
     8461 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     8521 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
     8581 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     8641 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     8701 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     8761 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     8821 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     8881 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     8941 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     9001 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     9061 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     9121 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     9181 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     9241 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     9301 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     9361 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     9421 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     9481 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     9541 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     9601 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     9661 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     9721 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     9781 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     9841 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     9901 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     9961 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
    10021 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
    10081 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
    10141 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
    10201 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
    10261 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
    10321 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
    10381 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
    10441 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
    10501 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
    10561 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
    10621 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
    10681 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
    10741 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
    10801 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
    10861 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
    10921 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
    10981 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
    11041 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
    11101 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
    11161 aagggcggtt t
//