LOCUS       sequence-204462-        9265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient expression of dCas9-VP64 fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9265)
  AUTHORS   Stephan Beck
  TITLE     Beck lab Cas9 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9265)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: p-dCas9-VP64-Hygro
FEATURES             Location/Qualifiers
     source          1..9265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(23..522)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(71..90)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     464..487
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     enhancer        527..906
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        907..1110
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1061..1081
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1152..1171
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1152..1170
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1178..1187
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1184..4585,4586..4588,4589..5185,5186,5187..5189,
                     5190..5191,5192..5194,5195..5287)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1184..1210,1211..1213,1214..3700,3701..3703,
                     3704..5287)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5294..5314
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5321..5386
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5417..5566
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             5575..5592
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5615..5632)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5621..5845
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5916..5932)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5916..5932)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5929..5951)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5940..5956
                     /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(5964..5970,5971..5988,5989..5994))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6009..6030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6147..6164)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6318..6906)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6398..6417)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7077..7868,7869..7937))
                     /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     7700..7719
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7938..8042)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8093..8214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8104..8123)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     8158..8177
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(8245..9265,1..5))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(join(8245..9265,1..5))
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
ORIGIN
        1 ttcatggtgg cgggatgcag gtcgaaaggc ccggagatga ggaagaggag aacagcgcgg
       61 cagacgtgcg cttttgaagc gtgcagaatg ccgggcctcc ggaggacctt cgggcgcccg
      121 ccccgcccct gagcccgccc ctgagcccgc ccccggaccc accccttccc agcctctgag
      181 cccagaaagc gaaggagcaa agctgctatt ggccgctgcc ccaaaggcct acccgcttcc
      241 attgctcagc ggtgctgtcc atctgcacga gactagtgag acgtgctact tccatttgtc
      301 acgtcctgca cgacgcgagc tgcggggcgg gggggaactt cctgactagg ggaggagtag
      361 aaggtggcgc gaaggggcca ccaaagaacg gagccggttg gcgcctaccg gtggatgtgg
      421 aatgtgtgcg aggccagagg ccacttgtgt agcgccaagt gcccagcggg gctgctaaag
      481 cgcatgctcc agactgcctt gggaaaagcg cctcccctac ccggttgaca ttgattattg
      541 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      601 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      661 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      721 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      781 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      841 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      901 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      961 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     1021 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     1081 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagaga
     1141 tccgcggccg ctaatacgac tcactatagg gagagccgcc accatggata aaaagtattc
     1201 tattggttta gccatcggca ctaattccgt tggatgggct gtcataaccg atgaatacaa
     1261 agtaccttca aagaaattta aggtgttggg gaacacagac cgtcattcga ttaaaaagaa
     1321 tcttatcggt gccctcctat tcgatagtgg cgaaacggca gaggcgactc gcctgaaacg
     1381 aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata tgttacttac aagaaatttt
     1441 tagcaatgag atggccaaag ttgacgattc tttctttcac cgtttggaag agtccttcct
     1501 tgtcgaagag gacaagaaac atgaacggca ccccatcttt ggaaacatag tagatgaggt
     1561 ggcatatcat gaaaagtacc caacgattta tcacctcaga aaaaagctag ttgactcaac
     1621 tgataaagcg gacctgaggt taatctactt ggctcttgcc catatgataa agttccgtgg
     1681 gcactttctc attgagggtg atctaaatcc ggacaactcg gatgtcgaca aactgttcat
     1741 ccagttagta caaacctata atcagttgtt tgaagagaac cctataaatg caagtggcgt
     1801 ggatgcgaag gctattctta gcgcccgcct ctctaaatcc cgacggctag aaaacctgat
     1861 cgcacaatta cccggagaga agaaaaatgg gttgttcggt aaccttatag cgctctcact
     1921 aggcctgaca ccaaatttta agtcgaactt cgacttagct gaagatgcca aattgcagct
     1981 tagtaaggac acgtacgatg acgatctcga caatctactg gcacaaattg gagatcagta
     2041 tgcggactta tttttggctg ccaaaaacct tagcgatgca atcctcctat ctgacatact
     2101 gagagttaat actgagatta ccaaggcgcc gttatccgct tcaatgatca aaaggtacga
     2161 tgaacatcac caagacttga cacttctcaa ggccctagtc cgtcagcaac tgcctgagaa
     2221 atataaggaa atattctttg atcagtcgaa aaacgggtac gcaggttata ttgacggcgg
     2281 agcgagtcaa gaggaattct acaagtttat caaacccata ttagagaaga tggatgggac
     2341 ggaagagttg cttgtaaaac tcaatcgcga agatctactg cgaaagcagc ggactttcga
     2401 caacggtagc attccacatc aaatccactt aggcgaattg catgctatac ttagaaggca
     2461 ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa tcctaacctt
     2521 tcgcatacct tactatgtgg gacccctggc ccgagggaac tctcggttcg catggatgac
     2581 aagaaagtcc gaagaaacga ttactccatg gaattttgag gaagttgtcg ataaaggtgc
     2641 gtcagctcaa tcgttcatcg agaggatgac caactttgac aagaatttac cgaacgaaaa
     2701 agtattgcct aagcacagtt tactttacga gtatttcaca gtgtacaatg aactcacgaa
     2761 agttaagtat gtcactgagg gcatgcgtaa acccgccttt ctaagcggag aacagaagaa
     2821 agcaatagta gatctgttat tcaagaccaa ccgcaaagtg acagttaagc aattgaaaga
     2881 ggactacttt aagaaaattg aatgcttcga ttctgtcgag atctccgggg tagaagatcg
     2941 atttaatgcg tcacttggta cgtatcatga cctcctaaag ataattaaag ataaggactt
     3001 cctggataac gaagagaatg aagatatctt agaagatata gtgttgactc ttaccctctt
     3061 tgaagatcgg gaaatgattg aggaaagact aaaaacatac gctcacctgt tcgacgataa
     3121 ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg ggacgattgt cgcggaaact
     3181 tatcaacggg ataagagaca agcaaagtgg taaaactatt ctcgattttc taaagagcga
     3241 cggcttcgcc aataggaact ttatgcagct gatccatgat gactctttaa ccttcaaaga
     3301 ggatatacaa aaggcacagg tttccggaca aggggactca ttgcacgaac atattgcgaa
     3361 tcttgctggt tcgccagcca tcaaaaaggg catactccag acagtcaaag tagtggatga
     3421 gctagttaag gtcatgggac gtcacaaacc ggaaaacatt gtaatcgaga tggcacgcga
     3481 aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag cggatgaaga gaatagaaga
     3541 gggtattaaa gaactgggca gccagatctt aaaggagcat cctgtggaaa atacccaatt
     3601 gcagaacgag aaactttacc tctattacct acaaaatgga agggacatgt atgttgatca
     3661 ggaactggac ataaaccgtt tatctgatta cgacgtcgat gccattgtac cccaatcctt
     3721 tttgaaggac gattcaatcg acaataaagt gcttacacgc tcggataaga accgagggaa
     3781 aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg aagaactatt ggcggcagct
     3841 cctaaatgcg aaactgataa cgcaaagaaa gttcgataac ttaactaaag ctgagagggg
     3901 tggcttgtct gaacttgaca aggccggatt tattaaacgt cagctcgtgg aaacccgcca
     3961 aatcacaaag catgttgcac agatactaga ttcccgaatg aatacgaaat acgacgagaa
     4021 cgataagctg attcgggaag tcaaagtaat cactttaaag tcaaaattgg tgtcggactt
     4081 cagaaaggat tttcaattct ataaagttag ggagataaat aactaccacc atgcgcacga
     4141 cgcttatctt aatgccgtcg tagggaccgc actcattaag aaatacccga agctagaaag
     4201 tgagtttgtg tatggtgatt acaaagttta tgacgtccgt aagatgatcg cgaaaagcga
     4261 acaggagata ggcaaggcta cagccaaata cttcttttat tctaacatta tgaatttctt
     4321 taagacggaa atcactctgg caaacggaga gatacgcaaa cgacctttaa ttgaaaccaa
     4381 tggggagaca ggtgaaatcg tatgggataa gggccgggac ttcgcgacgg tgagaaaagt
     4441 tttgtccatg ccccaagtca acatagtaaa gaaaactgag gtgcagaccg gagggttttc
     4501 aaaggaatcg attcttccaa aaaggaatag tgataagctc atcgctcgta aaaaggactg
     4561 ggacccgaaa aagtacggtg gcttcgatag ccctacagtt gcctattctg tcctagtagt
     4621 ggcaaaagtt gagaagggaa aatccaagaa actgaagtca gtcaaagaat tattggggat
     4681 aacgattatg gagcgctcgt cttttgaaaa gaaccccatc gacttccttg aggcgaaagg
     4741 ttacaaggaa gtaaaaaagg atctcataat taaactacca aagtatagtc tgtttgagtt
     4801 agaaaatggc cgaaaacgga tgttggctag cgccggagag cttcaaaagg ggaacgaact
     4861 cgcactaccg tctaaatacg tgaatttcct gtatttagcg tcccattacg agaagttgaa
     4921 aggttcacct gaagataacg aacagaagca actttttgtt gagcagcaca aacattatct
     4981 cgacgaaatc atagagcaaa tttcggaatt cagtaagaga gtcatcctag ctgatgccaa
     5041 tctggacaaa gtattaagcg catacaacaa gcacagggat aaacccatac gtgagcaggc
     5101 ggaaaatatt atccatttgt ttactcttac caacctcggc gctccagccg cattcaagta
     5161 ttttgacaca acgatagatc gcaaacgata cacttctacc aaggaggtgc tagacgcgac
     5221 actgattcac caatccatca cgggattata tgaaactcgg atagatttgt cacagcttgg
     5281 gggtgacgga tcccccaaga agaagaggaa agtctcgagc gactacaaag accatgacgg
     5341 tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaaggctg caggaggcgg
     5401 tggaagcggg cgcgccgacg cgctggacga tttcgatctc gacatgctgg gttctgatgc
     5461 cctcgatgac tttgacctgg atatgttggg aagcgacgca ttggatgact ttgatctgga
     5521 catgctcggc tccgatgctc tggacgattt cgatctcgat atgttataac cggtcatcat
     5581 caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     5641 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5701 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5761 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     5821 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata
     5881 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5941 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag
     6001 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     6061 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     6121 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     6181 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     6241 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     6301 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6361 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6421 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6481 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6541 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6601 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6661 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6721 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6781 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6841 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6901 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6961 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     7021 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     7081 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     7141 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     7201 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     7261 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     7321 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7381 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7441 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7501 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7561 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7621 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7681 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7741 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7801 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7861 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7921 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7981 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     8041 cgcacatttc cccgaaaagt gccacctgac gtcgacgggt atacagacat gataagatac
     8101 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     8161 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg
     8221 ggcgaagaac tctcggcatc tactctattc ctttgccctc ggacgagtgc tggggcgtcg
     8281 gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg
     8341 ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc
     8401 ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag
     8461 accaatgcgg agcatatacg cccggagccg cggcgatcct gcaagctccg gatgcctccg
     8521 ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt
     8581 ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat
     8641 gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac
     8701 ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact
     8761 gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat
     8821 gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct
     8881 cgtctggcta agatcggccg cagcgatcgc atccatggcc tccgcgaccg gctgcagaac
     8941 agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat
     9001 gcaataggtc aggctctcgc tgaattcccc aatgtcaagc acttccggaa tcgggagcgc
     9061 ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt
     9121 tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc
     9181 ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac
     9241 agacgtcgcg gtgagttcag gcttt
//
