LOCUS       sequence-79377-e       11589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized Cas9 protein and puromycin 
            resistance from EFS promoter and an EGFP targeting synthetic 
            single-guide RNA (sgRNA) element from U6 promoter. Lentiviral 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11589)
  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 11589)
  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.1247005"; journalName: "Science"; date: "2014-01-3-
            3"; volume: "343"; issue: "6166"; pages: "84-7"
COMMENT     Sequence Label: lentiCRISPR - EGFP sgRNA 2
FEATURES             Location/Qualifiers
     source          1..11589
                     /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        1986..2061
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2109..2226
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2295..2506
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2526..2535
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2535..2558
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2565..2585
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2610..6710
                     /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             2610..6710
                     /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             6711..6758
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6774..6830
                     /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             6831..7430
                     /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(6831..6850)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7327..7347
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7446..8034
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7499..7519)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7917..7928)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8106..8339
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8417..8538
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8454..8473)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8508..8527
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8578..8713
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8733..8752)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8734..8752)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8762..8779)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8762..8778)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8771..8793)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8920..9375
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9007..9026)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9217..9238
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9401..9505
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9506..9574,9575..10366)
                     /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(9724..9743)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10537..11125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11026..11045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11279..11296
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11413..11434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11449..11454,11455..11472,11473..11479)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11487..11503
                     /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     11492..11514
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11511..11527
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11511..11527
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11546..11566
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11548..11566
                     /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 acaccgagct ggacggcgac
     1981 gtaaagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     2041 aaagtggcac cgagtcggtg cttttttgaa ttctagatct tgagacaaat ggcagtattc
     2101 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     2161 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     2221 aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct cgagtggctc
     2281 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     2341 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     2401 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     2461 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg
     2521 gatccgccac catggattac aaagacgatg acgataagat ggccccaaag aagaagcgga
     2581 aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg
     2641 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     2701 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     2761 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     2821 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     2881 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     2941 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     3001 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     3061 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     3121 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     3181 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     3241 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     3301 agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact
     3361 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     3421 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     3481 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     3541 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     3601 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     3661 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     3721 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     3781 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     3841 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     3901 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     3961 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     4021 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     4081 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     4141 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     4201 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     4261 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     4321 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     4381 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     4441 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     4501 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     4561 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     4621 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     4681 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     4741 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     4801 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     4861 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     4921 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     4981 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     5041 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     5101 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     5161 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     5221 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     5281 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     5341 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     5401 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     5461 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     5521 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     5581 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     5641 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     5701 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     5761 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     5821 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     5881 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     5941 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     6001 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     6061 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     6121 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     6181 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     6241 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     6301 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     6361 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     6421 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     6481 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     6541 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     6601 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     6661 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgtcctg
     6721 ctgctactaa gaaagctggt caagctaaga aaaagaaagc tagcggcagc ggcgccacca
     6781 acttcagcct gctgaagcag gccggcgacg tggaggagaa ccccggcccc atgaccgagt
     6841 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     6901 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     6961 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     7021 tgtgggtcgc ggacgacggc gcggccgtgg cggtctggac cacgccggag agcgtcgaag
     7081 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     7141 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     7201 tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     7261 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     7321 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     7381 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag
     7441 tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     7501 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     7561 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     7621 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     7681 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     7741 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     7801 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     7861 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     7921 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     7981 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac
     8041 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     8101 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
     8161 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     8221 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     8281 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     8341 tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat
     8401 atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     8461 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     8521 actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc
     8581 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     8641 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     8701 ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     8761 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     8821 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     8881 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     8941 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     9001 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     9061 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     9121 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     9181 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     9241 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     9301 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     9361 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     9421 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     9481 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     9541 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     9601 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     9661 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     9721 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     9781 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     9841 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     9901 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     9961 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
    10021 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
    10081 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
    10141 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
    10201 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
    10261 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
    10321 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
    10381 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
    10441 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
    10501 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
    10561 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
    10621 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
    10681 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
    10741 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
    10801 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
    10861 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
    10921 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
    10981 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
    11041 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
    11101 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
    11161 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
    11221 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
    11281 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
    11341 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
    11401 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
    11461 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
    11521 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
    11581 gctgcaagc
//
