LOCUS       sequence-374870-       14558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human lentiviral vector for expression of dCas9 with a C-terminal 
            HA-2xNLS and GFP linked by a P2A site from a SFFV promoter with an 
            upstream ubiquitous chromatin opening element.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14558)
  AUTHORS   Replogle JM, Bonnar JL, Pogson AN, Liem CR, Maier NK, Ding Y, 
            Russell BJ, Wang X, Leng K, Guna A, Norman TM, Pak RA, Ramos DM, 
            Ward ME, Gilbert LA, Kampmann M, Weissman JS, Jost M
  TITLE     Maximizing CRISPRi efficacy and accessibility with dual-sgRNA 
            libraries and optimal effectors.
  JOURNAL   eLife. 2022 Dec 28;11:e81856. doi: 10.7554/eLife.81856.
  PUBMED    36576240
REFERENCE   2  (bases 1 to 14558)
  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"; journalName: "eLife."; 
            date: "2022-12-28"; volume: "11:e81856. doi"; pages: " 
            10.7554/eLife.81856"
COMMENT     Sequence Label: pHR-UCOE-SFFV-dCas9-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..14558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(25..43)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(26..43)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(333..352)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(566..585)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     685..707
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(745..763)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        831..935
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(936..1004,1005..1796)
                     /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(1154..1173)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1967..2555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2456..2475
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2709..2726
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2796..2816)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2801..3130
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2981..3116
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4264..4329
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4459..4479
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4904..5038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4941..4960)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4995..5014
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5206..5839
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5886..6011
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6508..6741
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6926..6970
                     /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"
     misc_feature    7255..7372
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8114..8521
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      8590..8599
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8596..8622,8623..8625,8626..11112,11113..11115,
                     11116..12699)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             8596..12699
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             12703..12729
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             12709..12735
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             12748..12768
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12775..12795
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12805..12861
                     /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             join(12862..12864,12865..13578)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(13165..13184)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     13512..13533
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    13627..14215
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(13680..13700)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(14098..14109)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(14218..14234)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14219..14235)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             14325..14558
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 gcatctagaa ttaattccgt gtattctata gtgtcaccta aatcgtatgt gtatgataca
       61 taaggttatg tattaattgt agccgcgttc taacgacaat atgtacaagc ctaattgtgt
      121 agcatctggc ttactgaagc agaccctatc atctctctcg taaactgccg tcagagtcgg
      181 tttggttgga cgaaccttct gagtttctgg taacgccgtc ccgcacccgg aaatggtcag
      241 cgaaccaatc agcagggtca tcgctagcca gatcctctac gccggacgca tcgtggccgg
      301 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
      361 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
      421 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
      481 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
      541 agagcgtcga atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
      601 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      661 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
      721 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
      781 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
      841 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
      901 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
      961 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1021 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1081 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1141 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     1201 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1261 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1321 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1381 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1441 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     1501 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1561 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1621 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1681 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1741 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1801 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     1861 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     1921 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1981 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2041 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2101 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2161 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2221 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2281 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2341 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2401 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2461 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2521 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2581 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2641 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2701 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     2761 tccccgcgcg ttggccgatt cattaatgca gctgtggaat gtgtgtcagt tagggtgtgg
     2821 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2881 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2941 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3001 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3061 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3121 cttttgcaaa aagcttggac acaagacagg cttgcgagat atgtttgaga ataccacttt
     3181 atcccgcgtc agggagaggc agtgcgtaaa aagacgcgga ctcatgtgaa atactggttt
     3241 ttagtgcgcc agatctctat aatctcgcgc aacctatttt cccctcgaac actttttaag
     3301 ccgtagataa acaggctggg acacttcaca tgagcgaaaa atacatcgtc acctgggaca
     3361 tgttgcagat ccatgcacgt aaactcgcaa gccgactgat gccttctgaa caatggaaag
     3421 gcattattgc cgtaagccgt ggcggtctgt accgggtgcg ttactggcgc gtgaactggg
     3481 tattcgtcat gtcgataccg tttgtatttc cagctacgat cacgacaacc agcgcgagct
     3541 taaagtgctg aaacgcgcag aaggcgatgg cgaaggcttc atcgttattg atgacctggt
     3601 ggataccggt ggtactgcgg ttgcgattcg tgaaatgtat ccaaaagcgc actttgtcac
     3661 catcttcgca aaaccggctg gtcgtccgct ggttgatgac tatgttgttg atatcccgca
     3721 agatacctgg attgaacagc cgtgggatat gggcgtcgta ttcgtcccgc caatctccgg
     3781 tcgctaatct tttcaacgcc tggcactgcc gggcgttgtt ctttttaact tcaggcgggt
     3841 tacaatagtt tccagtaagt attctggagg ctgcatccat gacacaggca aacctgagcg
     3901 aaaccctgtt caaaccccgc tttaaacatc ctgaaacctc gacgctagtc cgccgcttta
     3961 atcacggcgc acaaccgcct gtgcagtcgg cccttgatgg taaaaccatc cctcactggt
     4021 atcgcatgat taaccgtctg atgtggatct ggcgcggcat tgacccacgc gaaatcctcg
     4081 acgtccaggc acgtattgtg atgagcgatg ccgaacgtac cgacgatgat ttatacgata
     4141 cggtgattgg ctaccgtggc ggcaactgga tttatgagtg ggccccggat ctttgtgaag
     4201 gaaccttact tctgtggtgt gacataattg gacaaactac ctacagagat ttaaagctct
     4261 aaggtaaata taaaattttt aagtgtataa tgtgttaaac tactgattct aattgtttgt
     4321 gtattttaga ttccaaccta tggaactgat gaatgggagc agtggtggaa tgcctttaat
     4381 gaggaaaacc tgttttgctc agaagaaatg ccatctagtg atgatgaggc tactgctgac
     4441 tctcaacatt ctactcctcc aaaaaagaag agaaaggtag aagaccccaa ggactttcct
     4501 tcagaattgc taagtttttt gagtcatgct gtgtttagta atagaactct tgcttgcttt
     4561 gctatttaca ccacaaagga aaaagctgca ctgctataca agaaaattat ggaaaaatat
     4621 tctgtaacct ttataagtag gcataacagt tataatcata acatactgtt ttttcttact
     4681 ccacacaggc atagagtgtc tgctattaat aactatgctc aaaaattgtg tacctttagc
     4741 tttttaattt gtaaaggggt taataaggaa tatttgatgt atagtgcctt gactagagat
     4801 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     4861 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     4921 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4981 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcaa
     5041 ctggataact caagctaacc aaaatcatcc caaacttccc accccatacc ctattaccac
     5101 tgccaattac ctgtggtttc atttactcta aacctgtgat tcctctgaat tattttcatt
     5161 ttaaagaaat tgtatttgtt aaatatgtac tacaaactta gtagttggaa gggctaattc
     5221 actcccaaag aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc
     5281 ctgattagca gaactacaca ccagggccag gggtcagata tccactgacc tttggatggt
     5341 gctacaagct agtaccagtt gagccagata aggtagaaga ggccaataaa ggagagaaca
     5401 ccagcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtgttag
     5461 agtggaggtt tgacagccgc ctagcatttc atcacgtggc ccgagagctg catccggagt
     5521 acttcaagaa ctgctgatat cgagcttgct acaagggact ttccgctggg gactttccag
     5581 ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatcctgc atataagcag
     5641 ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     5701 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     5761 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     5821 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     5881 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     5941 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     6001 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     6061 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     6121 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     6181 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     6241 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     6301 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct
     6361 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     6421 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     6481 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     6541 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     6601 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     6661 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     6721 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     6781 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     6841 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     6901 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     6961 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     7021 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     7081 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     7141 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat
     7201 tcgattagtg aacggatctc gacggtatcg ccaaatggca gtattcatcc acaattttaa
     7261 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     7321 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     7381 ttacagggac agcagagatc cagtttggat cgataagctt gatatcgaat tcgggaggtg
     7441 gtccctgcag ttacgccaat gataaccccc gccagaaaaa tcttagtagc cttccctttt
     7501 tgttttccgt gccccaactc ggcggattga ctcggcccct tccggaaaca cccgaatcaa
     7561 cttctagtca aattattgtt cacgccgcaa tgacccaccc ctggcccgcg tctgtggaac
     7621 tgacccctgg tgtacaggag agttcgctgc tgaaagtggt cccaaagggg tactagtttt
     7681 taagctccca actccccctc ccccagcgtc tggaggattc cacaccctcg caccgcaggg
     7741 gcgaggaagt gggcggagtc cggttttggc gccagccgct gaggctgcca agcagaaaag
     7801 ccaccgctga ggagactccg gtcactgtcc tcgccccgcc tcccccttcc ctccccttgg
     7861 ggaccaccgg gcgccacgcc gcgaacggta agtgccgcgg tcgtcggcgc ctccgccctc
     7921 cccctagggc cccaattccc agcgggcgcg gcgcgcggcc ccgcgcgcag ctcccggctc
     7981 cctccccctt cggatgtggc ttgagctgta ggcgcggagt cctgcagccc cgataaaata
     8041 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     8101 caagctagct gcagtaacgc cattttgcaa ggcatggaaa aataccaaac caagaataga
     8161 gaagttcaga tcaagggcgg gtacatgaaa atagctaacg ttgggccaaa caggatatct
     8221 gcggtgagca gtttcggccc cggcccgggg ccaagaacag atggtcaccg cagtttcggc
     8281 cccggcccga ggccaagaac agatggtccc cagatatggc ccaaccctca gcagtttctt
     8341 aagacccatc agatgtttcc aggctccccc aaggacctga aatgaccctg cgccttattt
     8401 gaattaacca atcagcctgc ttctcgcttc tgttcgcgcg cttctgcttc ccgagctcta
     8461 taaaagagct cacaacccct cactcggcgc gccagtcctc cgacagactg agtcgcccgg
     8521 gggggatctg gagctctcga gaattctcac gcgtctgcag gatatcaagc ttgcggtacc
     8581 gcgggcccgg ccaccatgga caagaagtac agcatcggcc tggccatcgg caccaactct
     8641 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     8701 ggcaacaccg accggcacag catcaagaag aacctgatcg gcgccctgct gttcgacagc
     8761 ggagaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     8821 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     8881 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     8941 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     9001 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     9061 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     9121 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     9181 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     9241 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     9301 ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     9361 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     9421 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     9481 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     9541 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     9601 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     9661 aagaacggct acgccggcta catcgatggc ggagccagcc aggaagagtt ctacaagttc
     9721 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     9781 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     9841 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     9901 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     9961 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
    10021 tggaacttcg aggaagtggt ggacaagggc gccagcgccc agagcttcat cgagcggatg
    10081 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
    10141 gagtacttca ccgtgtacaa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
    10201 aagcccgcct tcctgagcgg cgagcagaaa aaagccatcg tggacctgct gttcaagacc
    10261 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
    10321 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
    10381 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
    10441 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
    10501 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
    10561 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
    10621 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
    10681 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
    10741 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
    10801 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
    10861 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
    10921 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
    10981 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
    11041 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
    11101 tacgatgtgg acgctatcgt gcctcagagc tttctgaagg acgactccat cgataacaaa
    11161 gtgctgactc ggagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
    11221 gtgaagaaga tgaagaacta ctggcgccag ctgctgaatg ccaagctgat tacccagagg
    11281 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
    11341 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
    11401 gactcccgga tgaacactaa gtacgacgag aacgacaaac tgatccggga agtgaaagtg
    11461 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
    11521 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
    11581 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
    11641 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
    11701 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
    11761 gagatccgga agcggcctct gatcgagaca aacggcgaaa caggcgagat cgtgtgggat
    11821 aagggccggg actttgccac cgtgcggaaa gtgctgtcta tgccccaagt gaatatcgtg
    11881 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
    11941 agcgacaagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
    12001 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
    12061 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
    12121 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
    12181 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
    12241 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
    12301 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
    12361 cagctgtttg tggaacagca caaacactac ctggacgaga tcatcgagca gatcagcgag
    12421 ttctccaaga gagtgatcct ggccgacgct aatctggaca aggtgctgag cgcctacaac
    12481 aagcacagag acaagcctat cagagagcag gccgagaata tcatccacct gtttaccctg
    12541 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
    12601 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
    12661 tacgagacac ggatcgacct gtctcagctg ggaggcgacg cctatcccta tgacgtgccc
    12721 gattatgcca gcctgggcag cggctccccc aagaaaaaac gcaaggtgga agatcctaag
    12781 aaaaagcgga aagtgagcgg ccgcgctact aacttcagcc tgctgaagca ggctggagac
    12841 gtggaggaga accctggacc tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
    12901 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc tggcgagggc
    12961 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
    13021 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
    13081 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
    13141 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
    13201 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
    13261 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
    13321 gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac
    13381 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
    13441 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
    13501 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
    13561 gacgagctgt acaagtaata agcgatcgcc ctgcaggcat gcaagcttga tatcaagctt
    13621 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
    13681 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
    13741 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
    13801 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
    13861 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
    13921 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
    13981 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
    14041 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
    14101 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
    14161 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
    14221 taccgtcgac ctcgagggaa ttaattcgag ctcggtacct ttaagaccaa tgacttacaa
    14281 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
    14341 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
    14401 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
    14461 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
    14521 cctcagaccc ttttagtcag tgtggaaaat ctctagca
//
