LOCUS       sequence-79438-e       13450 bp    DNA     circular SYN 14-APR-2025
DEFINITION  A genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080
            genes with 64,751 unique guide sequences enables both negative and 
            positive selection screening in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13450)
  AUTHORS   Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, 
            Heckl D, Ebert BL, Root DE, Doench JG, Zhang F
  TITLE     Genome-scale CRISPR-Cas9 knockout screening in human cells.
  JOURNAL   Science. 2014 Jan 3;343(6166):84-7. doi: 10.1126/science.1247005. 
            Epub 2013 Dec 12.
  PUBMED    24336571
REFERENCE   2  (bases 1 to 13450)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.1247005"; journalName: "Science"; date: "2014-01-3-
            3"; volume: "343"; issue: "6166"; pages: "84-7"
COMMENT     Sequence Label: Human GeCKO library
FEATURES             Location/Qualifiers
     source          1..13450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3847..3922
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3970..4087
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4156..4367
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4387..4396
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4396..4419
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4426..4446
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4471..8571
                     /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             4471..8571
                     /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             8572..8619
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8635..8691
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             8692..9291
                     /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(8692..8711)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9188..9208
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9307..9895
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9360..9380)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9778..9789)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9967..10200
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10278..10399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10315..10334)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10369..10388
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10439..10574
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10594..10613)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10595..10613)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10623..10640)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10623..10639)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10632..10654)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10781..11236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10868..10887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11078..11099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11262..11366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11367..11435,11436..12227)
                     /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(11585..11604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12398..12986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12887..12906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13140..13157
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13274..13295
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13310..13315,13316..13333,13334..13340)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13348..13364
                     /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     13353..13375
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13372..13388
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13372..13388
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13407..13427
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13409..13427
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cggttgtaaa
     1981 tgagcacaca aaatacacat gctaaaatat tatattctat gacctttata aaatcaacca
     2041 aaatcttctt tttaataact ttagtatcaa taattagaat ttttatgttc ctttttgcaa
     2101 acttttaata aaaatgagca aaataaaaaa acgctagttt tagtaactcg cgttgttttc
     2161 ttcaccttta ataatagcta ctccaccact tgttcctaag cggtcagctc ctgcttcaat
     2221 cattttttga gcatcttcaa atgttctaac tccaccagct gctttaacta aagcattgtc
     2281 tttaacaact gacttcatta gtttaacatc ttcaaatgtt gcacctgatt ttgaaaatcc
     2341 tgttgatgtt ttaacaaatt ctaatccagc ttcaacagct atttcacaag ctttcatgat
     2401 ttcttctttt gttaataaac aattttccat aatacattta acaacatgtg atccagctgc
     2461 tttttttaca gctttcatgt cttctaaaac taattcataa tttttgtctt ttaatgcacc
     2521 aatatttaat accatatcaa tttctgttgc accatcttta attgcttcag aaacttcgaa
     2581 tgcttttgta gctgttgtgc atgcacctag aggaaaacct acaacatttg ttattcctac
     2641 atttgtgcct tttaataatt ctttacaata gcttgttcaa tatgaattaa cacaaactgt
     2701 tgcaaaatca aattcaattg cttcatcaca taattgttta atttcagctt tcgtagcatc
     2761 ttgttttaat aatgtgtgat ctatatattt gtttagtttc attttttctc ctatatattc
     2821 atttttaatt ttaattcttt aataatttcg tctactttaa ctttagcgtt ttgaacagat
     2881 tcaccaacac ctataaaata aatttttagt ttaggttcag ttccacttgg gcgaacagca
     2941 aatcatgact tatcttctaa ataaaatttt agtaagtctt gtcctggcat attatacatt
     3001 ccatcgatgt agtcttcaac attaacaact ttaagtccag caatttgagt taagggtgtt
     3061 gctctcaatg atttcattaa tggttcaatt tttaatttct tttcttctgg tttaaaattc
     3121 aagtttaaag tgaaagtgta atatgcaccc atttctttaa ataaatcttc taaatagtct
     3181 actaatgttt tattttgttt tttataaaat caagcagcct ctgctattaa tatagaagct
     3241 tgtattccat ctttatctct agctgagtca tcaattacat atccataact ttcttcataa
     3301 gcaaaaacaa aatttaatcc gttatcttct tctttagcaa tttctctacc cattcattta
     3361 aatccagtta aagtttttac aatattaact ccatattttt catgagcgat tctatcaccc
     3421 aaatcacttg ttacaaaact tgaatataga gccggatttt ttggaatgct atttaagcgt
     3481 tttagatttg ataattttca atcaattaaa attggtcctg tttgatttcc atctaatctt
     3541 acaaaatgac catcatgttt tattgccatt ccaaatctgt cagcatctgg gtcattcata
     3601 ataataatat ctgcatcatg tttaatacca tattcaagcg gtatttttca tgcaggatca
     3661 aattctggat ttggatttac aacattttta aatgtttcat cttcaaatgc atgctcttca
     3721 acctcaataa cgttatatcc tgattcacgt aatatttttg gggtaaattt agttcctgtt
     3781 ccattaactg cgctaaaaat aatttttaaa tcttttttag cttcttgctc ttttttgtac
     3841 gtctctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     3901 aaaagtggca ccgagtcggt gcttttttga attctagatc ttgagacaaa tggcagtatt
     3961 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     4021 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     4081 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct
     4141 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     4201 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     4261 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     4321 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg
     4381 ggatccgcca ccatggatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
     4441 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
     4501 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     4561 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     4621 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     4681 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     4741 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     4801 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     4861 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     4921 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     4981 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     5041 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     5101 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     5161 gagaagaaga atggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac
     5221 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     5281 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     5341 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     5401 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     5461 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     5521 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     5581 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     5641 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     5701 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     5761 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     5821 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     5881 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     5941 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     6001 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     6061 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     6121 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     6181 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     6241 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     6301 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     6361 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     6421 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     6481 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     6541 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     6601 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     6661 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     6721 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     6781 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     6841 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     6901 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     6961 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     7021 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     7081 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     7141 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     7201 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     7261 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     7321 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     7381 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     7441 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     7501 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     7561 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     7621 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     7681 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     7741 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     7801 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     7861 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     7921 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     7981 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     8041 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     8101 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     8161 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     8221 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     8281 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     8341 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     8401 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     8461 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     8521 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caagcgtcct
     8581 gctgctacta agaaagctgg tcaagctaag aaaaagaaag ctagcggcag cggcgccacc
     8641 aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc catgaccgag
     8701 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     8761 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     8821 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     8881 gtgtgggtcg cggacgacgg cgcggccgtg gcggtctgga ccacgccgga gagcgtcgaa
     8941 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     9001 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     9061 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     9121 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     9181 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     9241 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     9301 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     9361 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     9421 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     9481 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     9541 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     9601 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     9661 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     9721 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     9781 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     9841 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     9901 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     9961 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
    10021 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
    10081 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
    10141 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
    10201 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
    10261 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
    10321 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
    10381 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
    10441 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
    10501 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
    10561 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
    10621 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
    10681 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
    10741 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
    10801 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
    10861 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
    10921 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
    10981 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
    11041 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
    11101 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
    11161 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
    11221 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
    11281 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
    11341 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
    11401 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
    11461 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
    11521 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
    11581 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
    11641 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
    11701 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
    11761 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
    11821 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
    11881 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
    11941 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
    12001 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
    12061 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
    12121 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
    12181 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
    12241 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
    12301 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
    12361 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
    12421 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
    12481 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
    12541 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
    12601 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
    12661 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
    12721 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
    12781 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
    12841 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
    12901 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
    12961 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
    13021 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
    13081 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
    13141 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
    13201 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
    13261 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
    13321 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
    13381 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
    13441 agctgcaagc
//
