LOCUS       sequence-354313-       13132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression vector for dCas9-VP64 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13132)
  AUTHORS   Schmidt R, Steinhart Z, Layeghi M, Freimer JW, Bueno R, Nguyen VQ, 
            Blaeschke F, Ye CJ, Marson A
  TITLE     CRISPR activation and interference screens decode stimulation 
            responses in primary human T cells.
  JOURNAL   Science. 2022 Feb 4;375(6580):eabj4008. doi: 
            10.1126/science.abj4008. Epub 2022 Feb 4.
  PUBMED    35113687
REFERENCE   2  (bases 1 to 13132)
  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: "Science."; 
            date: "2022-02-4"; volume: "375(6580):eabj4008. doi"; pages: " 
            10.1126/science.abj4008. Epub 2022 Feb 4"
COMMENT     Sequence Label: pZR112_Lenti-SFFV-mCherry-2A-dCas9-VP64
FEATURES             Location/Qualifiers
     source          1..13132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             23..203
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    250..375
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    868..1101
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1286..1330
                     /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    1628..1745
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1796..2203
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2210..2219
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2216..2923
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2361..2379)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2629..2648
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2939..2995
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2996..3043
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3044..3067,3068..3070,3071..5626,5627..5629,
                     5630..7144)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7199..7219
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7244..7393
                     /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"
     misc_feature    7412..8000
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7465..7485)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7883..7894)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8072..8305
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8331..8348)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8337..8561
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8607..9035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8694..8713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8904..8925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9044..9064)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9049..9378
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9229..9364
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9291..9310
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9426..9473
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9492..9866
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    9996..10117
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10033..10052)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10087..10106
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10166..10182)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10166..10182)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10179..10201)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10190..10206
                     /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(10214..10220,10221..10238,10239..10244))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10259..10280
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10397..10414)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10568..11156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10648..10667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11327..12118,12119..12187))
                     /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     11950..11969
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12188..12292)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12367..12386)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12558..12937
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(12939..13132,1..5)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     13092..13112
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 agcagcgcgt tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct
       61 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
      121 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
      181 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
      241 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
      301 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
      361 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
      421 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
      481 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
      541 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
      601 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
      661 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct
      721 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
      781 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
      841 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
      901 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
      961 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     1021 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     1081 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     1141 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     1201 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     1261 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     1321 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     1381 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     1441 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     1501 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat
     1561 tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca
     1621 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     1681 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     1741 attttcgggt ttattacagg gacagcagag atccagtttg gttaattaag gtaccgtaac
     1801 gccattttgc aaggcatgga aaaataccaa accaagaata gagaagttca gatcaagggc
     1861 gggtacatga aaatagctaa cgttgggcca aacaggatat ctgcggtgag cagtttcggc
     1921 cccggcccgg ggccaagaac agatggtcac cgcagtttcg gccccggccc gaggccaaga
     1981 acagatggtc cccagatatg gcccaaccct cagcagtttc ttaagaccca tcagatgttt
     2041 ccaggctccc ccaaggacct gaaatgaccc tgcgccttat ttgaattaac caatcagcct
     2101 gcttctcgct tctgttcgcg cgcttctgct tcccgagctc tataaaagag ctcacaaccc
     2161 ctcactcggc gcgccagtcc tccgacagac tgagtcgccc gggcgtacgg ccaccatggt
     2221 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     2281 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     2341 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     2401 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     2461 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     2521 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     2581 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     2641 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     2701 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     2761 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     2821 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     2881 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagattaacg gatccggcgc
     2941 aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgaaaag
     3001 gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aaggacaaga agtacagcat
     3061 cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     3121 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     3181 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     3241 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     3301 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     3361 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     3421 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     3481 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     3541 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     3601 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     3661 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     3721 ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc tgagcctggg
     3781 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     3841 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     3901 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     3961 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     4021 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     4081 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     4141 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     4201 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     4261 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     4321 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     4381 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     4441 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     4501 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     4561 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     4621 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     4681 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     4741 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     4801 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     4861 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     4921 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     4981 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     5041 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     5101 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     5161 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     5221 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     5281 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     5341 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     5401 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     5461 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     5521 actggacatc aaccggctgt ccgactacga tgtggaccac atcgtgcctc agagctttct
     5581 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaaggccc ggggcaagag
     5641 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     5701 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     5761 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     5821 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     5881 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     5941 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     6001 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     6061 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     6121 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     6181 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     6241 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     6301 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     6361 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     6421 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     6481 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     6541 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     6601 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     6661 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     6721 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     6781 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     6841 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     6901 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     6961 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     7021 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     7081 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     7141 cgacagcgct ggaggaggtg gaagcggagg aggaggaagc ggaggaggag gtagcggacc
     7201 taagaaaaag aggaaggtgg cggccgctgg atccggacgg gctgacgcat tggacgattt
     7261 tgatctggat atgctgggaa gtgacgccct cgatgatttt gaccttgaca tgcttggttc
     7321 ggatgccctt gatgactttg acctcgacat gctcggcagt gacgcccttg atgatttcga
     7381 cctggacatg ctgtgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     7441 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     7501 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     7561 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     7621 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     7681 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     7741 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     7801 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     7861 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     7921 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     7981 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     8041 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     8101 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     8161 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     8221 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     8281 tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag cctcgactgt
     8341 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     8401 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     8461 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     8521 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     8581 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     8641 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     8701 cgctttcttc ccttcctttc tggccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     8761 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     8821 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     8881 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     8941 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     9001 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     9061 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     9121 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     9181 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     9241 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     9301 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     9361 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag
     9421 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
     9481 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
     9541 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
     9601 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
     9661 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
     9721 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
     9781 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
     9841 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct
     9901 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     9961 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
    10021 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
    10081 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
    10141 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
    10201 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
    10261 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
    10321 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
    10381 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
    10441 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
    10501 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
    10561 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
    10621 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
    10681 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
    10741 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
    10801 ttcgctccaa gctgggctgt gtgcatgaac cccccgttca gcccgaccgc tgcgccttat
    10861 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
    10921 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
    10981 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
    11041 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
    11101 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
    11161 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
    11221 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
    11281 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
    11341 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
    11401 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
    11461 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
    11521 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
    11581 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
    11641 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
    11701 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
    11761 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
    11821 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
    11881 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
    11941 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
    12001 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
    12061 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
    12121 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
    12181 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
    12241 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
    12301 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
    12361 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
    12421 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
    12481 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
    12541 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
    12601 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
    12661 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
    12721 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
    12781 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
    12841 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
    12901 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
    12961 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
    13021 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
    13081 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata ta
//
