LOCUS       sequence-413633-       12449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing doxycycline-inducible human 
            codon-optimized SpCas9(D10A)-6xNLS (Cas9 nickase).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12449)
  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 12449)
  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-NoABE
FEATURES             Location/Qualifiers
     source          1..12449
                     /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(863..886,887..889,890..4963)
                     /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             863..4963
                     /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             4985..5011
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5021..5047
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5057..5083
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5093..5119
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5129..5155
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             5165..5191
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      5201..5210
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        5210..5421
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             5437..5832
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             5839..5892
                     /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(5893..6510,6511..6516,6517..6552,6553..6555,6556..6591,
                     6592..6594,6595..6630,6631..6639)
                     /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    6655..7243
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6708..6728)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7126..7137)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7315..7548
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7626..7747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7663..7682)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7717..7736
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7787..7922
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7942..7961)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7943..7961)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7971..7988)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7971..7987)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7980..8002)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8129..8584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8216..8235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8426..8447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8610..8714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8715..8783,8784..9575)
                     /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(8933..8952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9746..10334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10235..10254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10488..10505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10622..10643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10658..10663,10664..10681,10682..10688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10696..10712
                     /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     10701..10723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10720..10736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10720..10736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10755..10775
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10757..10775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10803..11029
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             11030..11210
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11257..11382
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11875..12108
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12293..12337
                     /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 ctgacaagaa gtacagcatc ggcctggcca tcggcaccaa
      901 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
      961 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
     1021 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
     1081 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
     1141 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
     1201 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
     1261 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
     1321 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     1381 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     1441 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     1501 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     1561 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     1621 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
     1681 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     1741 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     1801 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
     1861 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     1921 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     1981 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     2041 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
     2101 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     2161 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     2221 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     2281 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     2341 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     2401 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     2461 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
     2521 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     2581 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     2641 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     2701 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     2761 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     2821 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
     2881 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
     2941 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
     3001 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
     3061 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
     3121 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
     3181 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
     3241 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta
     3301 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
     3361 cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa
     3421 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga
     3481 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca
     3541 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc
     3601 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat
     3661 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa
     3721 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa
     3781 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg
     3841 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa
     3901 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc
     3961 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     4021 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     4081 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     4141 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     4201 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     4261 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     4321 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     4381 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     4441 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     4501 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     4561 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     4621 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     4681 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     4741 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     4801 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     4861 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     4921 cctgtacgag acacggatcg acctgtctca gctgggaggt gacagcggcg ggaattccag
     4981 atcccctgct gctaaacgtg ttaagcttga tgggggtagc ccggcagcca agagagtcaa
     5041 actcgacggc ggctccccag cggcaaaaag ggtgaaacta gacgggggta gccccgccgc
     5101 gaagcgtgta aagctggatg gaggatcgcc tgcggctaag cgagtcaaat tagacggtgg
     5161 gtcaccggct gcaaagcgcg tcaaattgga ttgagctagc gccaccatgg ggcagagcgc
     5221 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag
     5281 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     5341 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     5401 gggtttgccg ccagaacaca gttcgaacaa ttcaccatgg ccaagccttt gtctcaagaa
     5461 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     5521 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     5581 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     5641 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     5701 ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg
     5761 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     5821 gtgtgggagg gcggtaccga aggtagaggt tctctcctca cttgtggtga tgttgaagaa
     5881 aaccctggtc caatgtctag actggacaag agcaaagtca taaacggagc tctggaatta
     5941 ctcaatggtg tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt
     6001 gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctgcca
     6061 atcgagatgc tggacaggca tcatacccac ttctgccccc tggaaggcga gtcatggcaa
     6121 gactttctgc ggaacaacgc caagtcatac cgctgtgctc tcctctcaca tcgcgacggg
     6181 gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag
     6241 ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc
     6301 gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt agcaaaagag
     6361 gaaagagaga cacctaccac cgattctatg cccccacttc tgagacaagc aattgagctg
     6421 ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc
     6481 ctggagaaac agctaaagtg cgaaagcggc gggccgaccg acgcccttga cgattttgac
     6541 ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac
     6601 gctcttgacg attttgacct tgacatgctc cccgggtaaa cgcgttaagt cgacaatcaa
     6661 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     6721 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     6781 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     6841 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     6901 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     6961 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     7021 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     7081 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     7141 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     7201 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa
     7261 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     7321 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     7381 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     7441 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     7501 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta
     7561 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     7621 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     7681 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     7741 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     7801 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     7861 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     7921 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     7981 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     8041 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     8101 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     8161 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     8221 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     8281 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     8341 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     8401 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     8461 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     8521 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     8581 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     8641 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     8701 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     8761 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     8821 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     8881 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     8941 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     9001 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     9061 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     9121 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     9181 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     9241 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     9301 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     9361 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     9421 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     9481 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     9541 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     9601 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     9661 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     9721 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     9781 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     9841 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     9901 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     9961 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
    10021 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
    10081 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
    10141 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
    10201 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
    10261 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
    10321 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
    10381 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
    10441 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
    10501 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
    10561 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
    10621 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
    10681 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
    10741 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt
    10801 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
    10861 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
    10921 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
    10981 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
    11041 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
    11101 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
    11161 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
    11221 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
    11281 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
    11341 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
    11401 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
    11461 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
    11521 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
    11581 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
    11641 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
    11701 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
    11761 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
    11821 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
    11881 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
    11941 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
    12001 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
    12061 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
    12121 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
    12181 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
    12241 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
    12301 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
    12361 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
    12421 agtttttgct gtactttcta tagtgaata
//
