LOCUS       sequence-255735-       13177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral plasmid encoding Cas9, TagBFP2, and a gRNA
            backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13177)
  AUTHORS   Giuliano CJ, Lin A, Girish V, Sheltzer JM
  TITLE     Generating Single Cell-Derived Knockout Clones in Mammalian Cells 
            with CRISPR/Cas9.
  JOURNAL   Curr Protoc Mol Biol. 2019 Sep;128(1):e100. doi: 10.1002/cpmb.100.
  PUBMED    31503414
REFERENCE   2  (bases 1 to 13177)
  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: "Curr Protoc
            Mol Biol."; date: "2019-09"; volume: "128(1):e100. doi"; pages: " 
            10.1002/cpmb.100"
COMMENT     Sequence Label: Lenti-Cas9-gRNA-TagBFP2
FEATURES             Location/Qualifiers
     source          1..13177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     promoter        complement(3349..3597)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3407..3426)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3577..3597)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3604..3721
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3790..4001
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4021..4030
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4030..4053
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4060..4080
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4105..8205
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4105..8205
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8206..8253
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8269..8325
                     /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             8326..9027
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    9034..9622
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9087..9107)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9505..9516)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9694..9927
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10005..10126
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10042..10061)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10096..10115
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10166..10301
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10321..10340)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10322..10340)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10350..10367)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10350..10366)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10359..10381)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10508..10963
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10595..10614)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10805..10826
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10989..11093
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11094..11162,11163..11954)
                     /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(11312..11331)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12125..12713
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12614..12633
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12867..12884
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13001..13022
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13037..13042,13043..13060,13061..13067)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13075..13091
                     /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)."
     primer_bind     13080..13102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13099..13115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13099..13115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13134..13154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13136..13154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcgc catttgtctc aagatctaga attcaaaaaa agcaccgact
     1381 cggtgccact ttgctgtttc cagcaaagtt gataacggac tagccttatt taaacttgct
     1441 atgctgtttc cagcatagct cttaaacaga gacgtacaaa aaagagcaag aagctaaaaa
     1501 agatttaaaa attattttta gcgcagttaa tggaacagga actaaattta ccccaaaaat
     1561 attacgtgaa tcaggatata acgttattga ggttgaagag catgcatttg aagatgaaac
     1621 atttaaaaat gttgtaaatc caaatccaga atttgatcct gcatgaaaaa taccgcttga
     1681 atatggtatt aaacatgatg cagatattat tattatgaat gacccagatg ctgacagatt
     1741 tggaatggca ataaaacatg atggtcattt tgtaagatta gatggaaatc aaacaggacc
     1801 aattttaatt gattgaaaat tatcaaatct aaaacgctta aatagcattc caaaaaatcc
     1861 ggctctatat tcaagttttg taacaagtga tttgggtgat agaatcgctc atgaaaaata
     1921 tggagttaat attgtaaaaa ctttaactgg atttaaatga atgggtagag aaattgctaa
     1981 agaagaagat aacggattaa attttgtttt tgcttatgaa gaaagttatg gatatgtaat
     2041 tgatgactca gctagagata aagatggaat acaagcttct atattaatag cagaggctgc
     2101 ttgattttat aaaaaacaaa ataaaacatt agtagactat ttagaagatt tatttaaaga
     2161 aatgggtgca tattacactt tcactttaaa cttgaatttt aaaccagaag aaaagaaatt
     2221 aaaaattgaa ccattaatga aatcattgag agcaacaccc ttaactcaaa ttgctggact
     2281 taaagttgtt aatgttgaag actacatcga tggaatgtat aatatgccag gacaagactt
     2341 actaaaattt tatttagaag ataagtcatg atttgctgtt cgcccaagtg gaactgaacc
     2401 taaactaaaa atttatttta taggtgttgg tgaatctgtt caaaacgcta aagttaaagt
     2461 agacgaaatt attaaagaat taaaattaaa aatgaatata taggagaaaa aatgaaacta
     2521 aacaaatata tagatcacac attattaaaa caagatgcta cgaaagctga aattaaacaa
     2581 ttatgtgatg aagcaattga atttgatttt gcaacagttt gtgttaattc atattgaaca
     2641 agctattgta aagaattatt aaaaggcaca aatgtaggaa taacaaatgt tgtaggtttt
     2701 cctctaggtg catgcacaac agctacaaaa gcattcgaag tttctgaagc aattaaagat
     2761 ggtgcaacag aaattgatat ggtattaaat attggtgcat taaaagacaa aaattatgaa
     2821 ttagttttag aagacatgaa agctgtaaaa aaagcagctg gatcacatgt tgttaaatgt
     2881 attatggaaa attgtttatt aacaaaagaa gaaatcatga aagcttgtga aatagctgtt
     2941 gaagctggat tagaatttgt taaaacatca acaggatttt caaaatcagg tgcaacattt
     3001 gaagatgtta aactaatgaa gtcagttgtt aaagacaatg ctttagttaa agcagctggt
     3061 ggagttagaa catttgaaga tgctcaaaaa atgattgaag caggagctga ccgcttagga
     3121 acaagtggtg gagtagctat tattaaaggt gaagaaaaca acgcgagtta ctaaaactag
     3181 cgttttttta ttttgctcat ttttattaaa agtttgcaaa aaggaacata aaaattctaa
     3241 ttattgatac taaagttatt aaaaagaaga ttttggttga ttttataaag gtcatagaat
     3301 ataatatttt agcatgtgta ttttgtgtgc tcatttacaa ccgtctccgg tgtttcgtcc
     3361 tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg
     3421 atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct
     3481 acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc
     3541 tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctccca
     3601 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     3661 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     3721 tcgggtttat tacagggaca gcagagatcc actttggcgc cggctcgagt ggctccggtg
     3781 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     3841 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     3901 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     3961 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cgcgggatcc
     4021 gccaccatgg attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc
     4081 ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc
     4141 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     4201 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     4261 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     4321 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     4381 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     4441 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     4501 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     4561 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     4621 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     4681 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     4741 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     4801 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     4861 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     4921 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     4981 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     5041 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     5101 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     5161 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     5221 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     5281 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     5341 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     5401 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     5461 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     5521 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     5581 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     5641 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     5701 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     5761 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     5821 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     5881 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     5941 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     6001 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     6061 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     6121 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     6181 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     6241 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     6301 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     6361 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     6421 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     6481 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     6541 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     6601 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     6661 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     6721 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     6781 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     6841 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     6901 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     6961 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     7021 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     7081 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     7141 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     7201 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     7261 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     7321 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     7381 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     7441 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     7501 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     7561 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     7621 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     7681 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     7741 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     7801 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     7861 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     7921 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     7981 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
     8041 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
     8101 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     8161 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaagcg tcctgctgct
     8221 actaagaaag ctggtcaagc taagaaaaag aaagctagcg gcagcggcgc caccaacttc
     8281 agcctgctga agcaggccgg cgacgtggag gagaaccccg gccccatgag cgagctgatt
     8341 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag
     8401 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg
     8461 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc
     8521 agcaagacct tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct
     8581 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     8641 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     8701 ttcacatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgaa
     8761 acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg
     8821 ggcgggagcc atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag
     8881 aacctcaaga tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc
     8941 aacaacgaga cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct
     9001 agcaaactgg ggcacaagct taattgagtc gacaatcaac ctctggatta caaaatttgt
     9061 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     9121 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     9181 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     9241 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     9301 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     9361 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     9421 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     9481 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     9541 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     9601 tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag
     9661 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     9721 gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg
     9781 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     9841 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     9901 tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc
     9961 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag
    10021 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
    10081 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct
    10141 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
    10201 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
    10261 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
    10321 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
    10381 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
    10441 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
    10501 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
    10561 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
    10621 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
    10681 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
    10741 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
    10801 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
    10861 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
    10921 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
    10981 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
    11041 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
    11101 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
    11161 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
    11221 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
    11281 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
    11341 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
    11401 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
    11461 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
    11521 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
    11581 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
    11641 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
    11701 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
    11761 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
    11821 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
    11881 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
    11941 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
    12001 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
    12061 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
    12121 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
    12181 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
    12241 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
    12301 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
    12361 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
    12421 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
    12481 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
    12541 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
    12601 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
    12661 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
    12721 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
    12781 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
    12841 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
    12901 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
    12961 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
    13021 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
    13081 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
    13141 accctcacta aagggaacaa aagctggagc tgcaagc
//
