LOCUS       sequence-413620-       13547 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing doxycycline-inducible human 
            codon-optimized SpCas9(D10A)-6xNLS (Cas9 nickase) linked to ssDNA 
            binding domain from RAD51 and uracil DNA glycosylase inhibitor 
            (UGI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13547)
  AUTHORS   Xiao MS, Damodaran AP, Kumari B, Dickson E, Xing K, On TA, Parab N, 
            King HE, Perez AR, Guiblet WM, Duncan G, Che A, Chari R, Andresson 
            T, Vidigal JA, Weatheritt RJ, Aregger M, Gonatopoulos-Pournatzis T
  TITLE     Genome-scale exon perturbation screens uncover exons critical for 
            cell fitness.
  JOURNAL   Mol Cell. 2024 Jul 11;84(13):2553-2572.e19. doi: 
            10.1016/j.molcel.2024.05.024. Epub 2024 Jun 24.
  PUBMED    38917794
REFERENCE   2  (bases 1 to 13547)
  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"; doi: 
            "10.1016/j.molcel.2024.05.024"; journalName: "Mol Cell"; date: 
            "2024-07-11- 11"; volume: "84"; issue: "13"; pages: "2553-2572.e19"
COMMENT     Sequence Label: pLenti-nSpCas9-NoCBE
FEATURES             Location/Qualifiers
     source          1..13547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..86
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    287..404
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        469..783
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    477..495
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    513..531
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    548..566
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    584..602
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    620..638
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    655..673
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    691..709
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     756..780
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             806..829
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             830..856
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1394..1417,1418..1420,1421..5494)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1394..5494
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5531..5779
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5810..6058
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6083..6109
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6119..6145
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6155..6181
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6191..6217
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6227..6253
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6263..6289
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      6299..6308
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        6308..6519
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             6535..6930
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             6937..6990
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6991..7608,7609..7614,7615..7650,7651..7653,7654..7689,
                     7690..7692,7693..7728,7729..7737)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    7753..8341
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7806..7826)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8224..8235)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8413..8646
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8724..8845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8761..8780)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8815..8834
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8885..9020
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9040..9059)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9041..9059)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9069..9086)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9069..9085)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9078..9100)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9227..9682
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9314..9333)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9524..9545
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9708..9812
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9813..9881,9882..10673)
                     /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(10031..10050)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10844..11432
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11333..11352
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11586..11603
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11720..11741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11756..11761,11762..11779,11780..11786)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11794..11810
                     /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     11799..11821
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11818..11834
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11818..11834
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11853..11873
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11855..11873
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        11901..12127
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             12128..12308
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    12355..12480
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12973..13206
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             13391..13435
                     /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"
ORIGIN
        1 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
       61 ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
      121 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      181 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      241 atcataaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
      301 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      361 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
      421 acagcagaga tccactttgg cgccggctcg agcgcgaatc gatatgtcga gtttactccc
      481 tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgatgtcg
      541 agtttactcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga
      601 gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttatcccta
      661 tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga
      721 ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc
      781 gcctggagaa ttggctagca ccatggatta caaagacgat gacgataagt acccatacga
      841 tgtgccagat tacgccagcg gtaccggatc tagcggagga tcaagcggaa gcgagactcc
      901 tggaaccagc gaaagcgcaa ccccagaaag cagcggagga agtagcggag gaagcatggc
      961 aatgcagatg cagcttgaag caaatgcaga tacttcagtg gaagaagaaa gctttggccc
     1021 acaacccatt tcacggttag agcagtgtgg cataaatgcc aacgatgtga agaaattgga
     1081 agaagctgga ttccatactg tggaggctgt tgcctatgcg ccaaagaagg agctaataaa
     1141 tattaaggga attagtgaag ccaaagctga taaaattctg gctgaggcag ctaaattagt
     1201 tccaatgggt ttcaccactg caactgaatt ccaccaaagg cggtcagaga tcatacagat
     1261 tactactggc tccaaagagc ttgacaaact acttcaatct ggcggatcta gcggtggatc
     1321 tagcggctct gagacccctg gaacatccga atccgccact ccagagagca gcggaggctc
     1381 ttctggaagc gctgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg
     1441 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     1501 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     1561 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     1621 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     1681 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     1741 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     1801 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     1861 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     1921 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     1981 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     2041 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     2101 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     2161 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     2221 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     2281 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     2341 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     2401 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     2461 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     2521 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     2581 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     2641 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     2701 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     2761 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     2821 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     2881 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     2941 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     3001 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     3061 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     3121 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     3181 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     3241 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     3301 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     3361 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     3421 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     3481 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     3541 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     3601 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     3661 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     3721 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     3781 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     3841 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     3901 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     3961 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     4021 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     4081 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     4141 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     4201 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     4261 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     4321 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     4381 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     4441 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     4501 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     4561 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     4621 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     4681 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
     4741 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
     4801 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     4861 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     4921 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     4981 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     5041 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     5101 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     5161 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     5221 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     5281 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     5341 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
     5401 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     5461 gacacggatc gacctgtctc agctgggagg tgacagtggc ggaagtggac tagtaggaag
     5521 tggcgggagc actaatctga gcgacatcat tgagaaggag actgggaaac agctggtcat
     5581 tcaggagtcc atcctgatgc tgcctgagga ggtggaggaa gtgatcggca acaagccaga
     5641 gtctgacatc ctggtgcaca ccgcctacga cgagtccaca gatgagaatg tgatgctgct
     5701 gacctctgac gcccccgagt ataagccttg ggccctggtc atccaggatt ctaacggcga
     5761 gaataagatc aagatgctga gcggaggatc cggaggatct ggaggcagca ccaacctgtc
     5821 tgacatcatc gagaaggaga caggcaagca gctggtcatc caggagagca tcctgatgct
     5881 gcccgaagaa gtcgaagaag tgatcggaaa caagcctgag agcgatatcc tggtccatac
     5941 cgcctacgac gagagtaccg acgaaaatgt gatgctgctg acatccgacg ccccagagta
     6001 taagccctgg gctctggtca tccaggattc caacggagag aacaaaatca aaatgctgtc
     6061 tggcggctca gaattcggat cccctgctgc taaacgtgtt aagcttgatg ggggtagccc
     6121 ggcagccaag agagtcaaac tcgacggcgg ctccccagcg gcaaaaaggg tgaaactaga
     6181 cgggggtagc cccgccgcga agcgtgtaaa gctggatgga ggatcgcctg cggctaagcg
     6241 agtcaaatta gacggtgggt caccggctgc aaagcgcgtc aaattggatt gagctagcgc
     6301 caccatgggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     6361 aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     6421 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     6481 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagt tcgaacaatt caccatggcc
     6541 aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc
     6601 atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc
     6661 ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg
     6721 ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag
     6781 aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct
     6841 gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa
     6901 ttgctgccct ctggttatgt gtgggagggc ggtaccgaag gtagaggttc tctcctcact
     6961 tgtggtgatg ttgaagaaaa ccctggtcca atgtctagac tggacaagag caaagtcata
     7021 aacggagctc tggaattact caatggtgtc ggtatcgaag gcctgacgac aaggaaactc
     7081 gctcaaaagc tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc
     7141 ctgctcgatg ccctgccaat cgagatgctg gacaggcatc atacccactt ctgccccctg
     7201 gaaggcgagt catggcaaga ctttctgcgg aacaacgcca agtcataccg ctgtgctctc
     7261 ctctcacatc gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac
     7321 gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca
     7381 ctgtacgctc tgtccgccgt gggccacttt acactgggct gcgtattgga ggaacaggag
     7441 catcaagtag caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg
     7501 agacaagcaa ttgagctgtt cgaccggcag ggagccgaac ctgccttcct tttcggcctg
     7561 gaactaatca tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccgaccgac
     7621 gcccttgacg attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt
     7681 gatatgctgc ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtaaacg
     7741 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     7801 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     7861 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     7921 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     7981 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     8041 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     8101 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     8161 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     8221 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     8281 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     8341 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     8401 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     8461 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     8521 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     8581 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     8641 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     8701 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     8761 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     8821 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     8881 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     8941 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     9001 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     9061 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     9121 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     9181 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     9241 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     9301 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     9361 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     9421 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     9481 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     9541 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     9601 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     9661 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     9721 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     9781 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     9841 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     9901 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     9961 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
    10021 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
    10081 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
    10141 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
    10201 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
    10261 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
    10321 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
    10381 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
    10441 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
    10501 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
    10561 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
    10621 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
    10681 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
    10741 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
    10801 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
    10861 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
    10921 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
    10981 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
    11041 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
    11101 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
    11161 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
    11221 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
    11281 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
    11341 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
    11401 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
    11461 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
    11521 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
    11581 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
    11641 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
    11701 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
    11761 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
    11821 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
    11881 agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt
    11941 aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt
    12001 ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat
    12061 tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata
    12121 cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
    12181 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
    12241 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
    12301 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
    12361 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
    12421 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
    12481 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
    12541 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
    12601 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
    12661 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
    12721 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
    12781 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
    12841 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
    12901 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
    12961 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
    13021 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
    13081 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
    13141 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
    13201 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
    13261 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
    13321 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
    13381 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
    13441 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
    13501 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaata
//
