LOCUS       sequence-140566-       11885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline-inducible lentiviral expression of SpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11885)
  AUTHORS   Wang T, Wei JJ, Sabatini DM, Lander ES
  TITLE     Genetic screens in human cells using the CRISPR-Cas9 system.
  JOURNAL   Science. 2014 Jan 3;343(6166):80-4. doi: 10.1126/science.1246981. 
            Epub 2013 Dec 12.
  PUBMED    24336569
REFERENCE   2  (bases 1 to 11885)
  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: 
            "10.1126/science.1246981"; journalName: "Science"; date: "2014-01-3-
            3"; volume: "343"; issue: "6166"; pages: "80-4"
COMMENT     Sequence Label: pCW-Cas9
FEATURES             Location/Qualifiers
     source          1..11885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        212..526
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    220..238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    256..274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    291..309
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    327..345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    363..381
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    398..416
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    434..452
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     499..523
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      543..552
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             552..617
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             624..644
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             669..4769
                     /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             669..4769
                     /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             4770..4817
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        4827..5337
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     5256..5274
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5347..5943
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5347..5366)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5843..5863
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5944..5997
                     /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(5998..6615,6616..6621,6622..6657,6658..6660,6661..6696,
                     6697..6699,6700..6735,6736..6744)
                     /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"
     primer_bind     complement(6784..6804)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7202..7213)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7418..7651
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7723..7844
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7760..7779)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7814..7833
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7884..8019
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        8019..8090
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8091..8159,8160..8951)
                     /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(8309..8328)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9122..9710
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9611..9630
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9864..9881
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9998..10019
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10034..10039,10040..10057,10058..10064)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10072..10088
                     /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     10077..10099
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10096..10112
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10096..10112
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10131..10151
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10133..10151
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10179..10405
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10406..10586
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    10633..10758
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11251..11484
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11669..11713
                     /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 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac
      241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
      301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
      421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
      481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta
      541 gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca
      601 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
      661 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
      721 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
      781 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
      841 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
      901 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
      961 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     1021 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     1081 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     1141 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     1201 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     1261 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     1321 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     1381 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     1441 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     1501 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     1561 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     1621 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     1681 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     1741 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     1801 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     1861 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     1921 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     1981 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     2041 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     2101 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     2161 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     2221 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     2281 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     2341 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     2401 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     2461 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     2521 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     2581 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     2641 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     2701 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     2761 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     2821 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     2881 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     2941 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     3001 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     3061 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     3121 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     3181 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     3241 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     3301 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     3361 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     3421 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     3481 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     3541 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     3601 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     3661 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     3721 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     3781 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     3841 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     3901 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     3961 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     4021 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     4081 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     4141 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     4201 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     4261 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     4321 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     4381 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     4441 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     4501 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     4561 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     4621 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     4681 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     4741 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
     4801 aggcaaaaaa gaaaaagtaa ggatccgggg ttggggttgc gccttttcca aggcagccct
     4861 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
     4921 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
     4981 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
     5041 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
     5101 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
     5161 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
     5221 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
     5281 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagcaa
     5341 ttcaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     5401 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     5461 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     5521 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     5581 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     5641 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     5701 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     5761 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     5821 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     5881 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     5941 gccgaaggta gaggttctct cctcacttgt ggtgatgttg aagaaaaccc tggtccaatg
     6001 tctagactgg acaagagcaa agtcataaac ggagctctgg aattactcaa tggtgtcggt
     6061 atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg
     6121 tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga gatgctggac
     6181 aggcatcata cccacttctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac
     6241 aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc
     6301 ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt
     6361 cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca
     6421 ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag agagacacct
     6481 accaccgatt ctatgccccc acttctgaga caagcaattg agctgttcga ccggcaggga
     6541 gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta
     6601 aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga catgctccca
     6661 gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt
     6721 gaccttgaca tgctccccgg gtaactaagt aaggatcgat ccaagatatc gtattcttaa
     6781 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     6841 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     6901 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     6961 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     7021 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     7081 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc
     7141 atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     7201 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     7261 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc
     7321 tgtttcgcct cggcgtccgg actagaggta cctttaagac caatgactta caaggcagct
     7381 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     7441 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     7501 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     7561 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     7621 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag
     7681 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     7741 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     7801 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     7861 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     7921 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     7981 agctattcca gaagtagtga ggaggctttt ttggaggcct ttcaaatatg tatccgctca
     8041 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     8101 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     8161 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     8221 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     8281 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     8341 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     8401 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     8461 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     8521 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     8581 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     8641 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     8701 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     8761 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     8821 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     8881 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     8941 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     9001 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     9061 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     9121 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     9181 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     9241 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     9301 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     9361 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     9421 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     9481 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     9541 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     9601 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     9661 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     9721 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     9781 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     9841 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     9901 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     9961 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
    10021 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
    10081 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
    10141 ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt
    10201 gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca
    10261 ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca
    10321 gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt
    10381 taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga
    10441 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
    10501 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
    10561 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
    10621 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
    10681 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
    10741 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
    10801 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
    10861 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
    10921 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
    10981 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
    11041 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
    11101 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
    11161 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
    11221 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
    11281 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
    11341 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
    11401 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
    11461 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
    11521 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
    11581 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
    11641 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
    11701 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
    11761 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
    11821 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
    11881 gggac
//
