LOCUS       sequence-226458-       12494 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Constitutive expression of Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12494)
  AUTHORS   Giacomelli AO, Yang X, Lintner RE, McFarland JM, Duby M, Kim J, 
            Howard TP, Takeda DY, Ly SH, Kim E, Gannon HS, Hurhula B, Sharpe T, 
            Goodale A, Fritchman B, Steelman S, Vazquez F, Tsherniak A, Aguirre 
            AJ, Doench JG, Piccioni F, Roberts CWM, Meyerson M, Getz G, 
            Johannessen CM, Root DE, Hahn WC
  TITLE     Mutational processes shape the landscape of TP53 mutations in human 
            cancer.
  JOURNAL   Nat Genet. 2018 Oct;50(10):1381-1387. doi: 
            10.1038/s41588-018-0204-y.
  PUBMED    30224644
REFERENCE   2  (bases 1 to 12494)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Nat Genet"; date: "2018-10"; volume: "50"; issue: "10"; pages: 
            "1381-1387"
COMMENT     Sequence Label: pLX311-Cas9
FEATURES             Location/Qualifiers
     source          1..12494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /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"
     promoter        1539..1749
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        1561..1757
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1608..1743
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1779..2174
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    2248..2365
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2503..3681
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2734..3672
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3629..3649
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    3701..3725
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3728..3793
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3800..3820
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3845..7945
                     /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             3845..7945
                     /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             7946..7993
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(7996..8020)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             8028..8069
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    8120..8708
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8173..8193)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8591..8602)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8780..9013
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9091..9212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9128..9147)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9182..9201
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9252..9387
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9407..9426)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9408..9426)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9436..9453)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9436..9452)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9445..9467)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9594..10049
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9681..9700)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9891..9912
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10075..10179
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10180..10248,10249..11040)
                     /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(10398..10417)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11211..11799
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11700..11719
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11953..11970
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12087..12108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12123..12128,12129..12146,12147..12153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12161..12177
                     /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     12166..12188
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12185..12201
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12185..12201
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     12220..12240
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        12222..12240
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        12268..12494
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccatgcattg catctcaatt
     1501 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     1561 tgcgtctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     1621 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     1681 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     1741 gcctaggctt ttgcaaaaag ctttctagag gtaccaccat ggccaagcct ttgtctcaag
     1801 aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag
     1861 actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg
     1921 tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg
     1981 cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga
     2041 gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag
     2101 tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt
     2161 atgtgtggga gggcctgcag ctgcagtagt aaggcgcgcc gttaacgaat tctagatctt
     2221 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2281 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2341 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
     2401 gcgccggctc gaggcctgca ggtgcaaaga tggataaagt tttaaacaga gaggaatctt
     2461 tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
     2521 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2581 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2641 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2701 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
     2761 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag
     2821 tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc
     2881 gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc
     2941 cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc
     3001 atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat
     3061 gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc
     3121 cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc
     3181 ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt
     3241 atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga
     3301 tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag
     3361 cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca
     3421 tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg
     3481 agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag
     3541 tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc
     3601 ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt
     3661 cttccatttc aggtgtcgtg aggctagcat cgattgatca acaagtttgt acaaaaaagt
     3721 tggcatggac tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga
     3781 cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc
     3841 agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt
     3901 gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg
     3961 gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga
     4021 ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg
     4081 ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag
     4141 actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg
     4201 caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa
     4261 gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca
     4321 catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga
     4381 cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc
     4441 catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag
     4501 acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa
     4561 cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga
     4621 ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc
     4681 ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat
     4741 cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc
     4801 tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg
     4861 gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc
     4921 cggctacatt gacggcggag ccagccagga agagttctac aagttcatca agcccatcct
     4981 ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg
     5041 gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca
     5101 cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat
     5161 cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag
     5221 cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga
     5281 agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa
     5341 gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt
     5401 gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct
     5461 gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac
     5521 cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat
     5581 ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat
     5641 tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt
     5701 gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc
     5761 ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg
     5821 caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct
     5881 ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga
     5941 cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct
     6001 gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac
     6061 agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt
     6121 gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag
     6181 aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc
     6241 cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg
     6301 ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca
     6361 tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag
     6421 cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa
     6481 gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct
     6541 gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca
     6601 gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa
     6661 cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc
     6721 caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa
     6781 ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa
     6841 gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa
     6901 gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag
     6961 caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg
     7021 gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt
     7081 tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt
     7141 gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat
     7201 cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc
     7261 ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt
     7321 gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga
     7381 ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa
     7441 gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact
     7501 gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag
     7561 ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga
     7621 acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt
     7681 gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa
     7741 gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc
     7801 ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa
     7861 agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat
     7921 cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc
     7981 aaaaaagaaa aagaccccca actttcttgt acaaagtggt tgatatcggt aagcctatcc
     8041 ctaaccctct cctcggtctc gattctacgt agtaatgaac tagtaccggt taagtcgaca
     8101 atcaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     8161 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     8221 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     8281 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     8341 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     8401 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     8461 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     8521 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     8581 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     8641 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     8701 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     8761 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     8821 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     8881 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     8941 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     9001 aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt
     9061 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     9121 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     9181 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     9241 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     9301 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     9361 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     9421 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     9481 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     9541 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     9601 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     9661 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     9721 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     9781 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     9841 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     9901 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     9961 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
    10021 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
    10081 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
    10141 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
    10201 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
    10261 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
    10321 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
    10381 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
    10441 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
    10501 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
    10561 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
    10621 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
    10681 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
    10741 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
    10801 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
    10861 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
    10921 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
    10981 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
    11041 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
    11101 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
    11161 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
    11221 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
    11281 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
    11341 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
    11401 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
    11461 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
    11521 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
    11581 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
    11641 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
    11701 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
    11761 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
    11821 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
    11881 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
    11941 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
    12001 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
    12061 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
    12121 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
    12181 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
    12241 gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca
    12301 acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc
    12361 gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga
    12421 catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag
    12481 ctcgatacat aaac
//
