LOCUS       sequence-412950-       15874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral Expression Plasmid for CRISPRoff2.1 driven by EF1a 
            Promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15874)
  AUTHORS   Allen TP, Roennfeldt AE, Reckdharajkumar M, Liu M, Quinn RJ, Russell
            DL, Peet DJ, Whitelaw ML, Bersten DC
  TITLE     dFLASH; dual FLuorescent transcription factor Activity Sensor for 
            Histone integrated live-cell reporting and high-content screening
  JOURNAL   bioRxiv 2023.11.21.568191
REFERENCE   2  (bases 1 to 15874)
  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: "bioRxiv 
            2023.11.21.568191"
COMMENT     Sequence Label: pLV-EF1a-CRISPRoffv2.1-IRES-BlastR
FEATURES             Location/Qualifiers
     source          1..15874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    159..180
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(195..200,201..218,219..225)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    233..249
                     /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     238..260
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     257..273
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     257..273
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     292..312
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        294..312
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        340..566
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             567..747
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    794..919
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1412..1645
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1830..1874
                     /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    2136..2253
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     complement(2256..2272)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2257..2273)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        2380..3566
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2611..3557
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3514..3534
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    3639..3663
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(5622..5648,5649..5651,5652..8138,8139..8141,
                     8142..9725)
                     /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             9729..9755
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             9735..9761
                     /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             9774..9794
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9801..9821
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9861..10556
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             10602..10787
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     protein_bind    complement(10817..10841)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    10986..11448
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(11042..11059)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     11269..11288
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             11452..11850
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             13083..13316
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    13388..13509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13425..13444)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13479..13498
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     13611..13630
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(13895..13913)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13981..14085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(14086..14154,14155..14946)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(14304..14323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      15117..15705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     15606..15625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg
       61 gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac
      121 cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat
      181 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      241 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      301 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
      361 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
      421 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
      481 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
      541 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
      601 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
      661 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
      721 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
      781 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
      841 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
      901 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
      961 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     1021 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     1081 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     1141 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     1201 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     1261 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     1321 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     1381 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     1441 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     1501 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     1561 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     1621 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     1681 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     1741 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     1801 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     1861 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     1921 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     1981 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     2041 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat
     2101 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg
     2161 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     2221 ttacaaaaac aaattacaaa aattcaaaat tttatcgata ccgtcgacct cgacggatcg
     2281 ggagatctcg accgagcttt gcaaagatgg ataaagtttt aaacagagag gaatctttgc
     2341 agctaatgga ccttctaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
     2401 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
     2461 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     2521 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     2581 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
     2641 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac
     2701 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
     2761 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
     2821 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
     2881 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
     2941 ggccaagatc gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
     3001 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
     3061 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     3121 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     3181 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     3241 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     3301 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgatcgagct
     3361 tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca
     3421 ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat
     3481 ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt
     3541 tttttcttcc atttcaggtg tcgtgaggaa ttagcttgac caagcttggt accgagctcg
     3601 gatccactag taacggccgc cagtgtgctg gaattatcac aagtttgtac aaaaaagcag
     3661 gctttaaagg aaccaattca gtcgactgga tccggtaccg aattctgcag tcgacggtac
     3721 cgcgggcccg ggatccaccg gtcgccacca tgaaccatga ccaggaattt gaccccccaa
     3781 aggtttaccc acctgtgcca gctgagaaga ggaagcccat ccgcgtgctg tctctctttg
     3841 atgggattgc tacagggctc ctggtgctga aggacctggg catccaagtg gaccgctaca
     3901 ttgcctccga ggtgtgtgag gactccatca cggtgggcat ggtgcggcac cagggaaaga
     3961 tcatgtacgt cggggacgtc cgcagcgtca cacagaagca tatccaggag tggggcccat
     4021 tcgacctggt gattggaggc agtccctgca atgacctctc cattgtcaac cctgcccgca
     4081 agggacttta tgagggtact ggccgcctct tctttgagtt ctaccgcctc ctgcatgatg
     4141 cgcggcccaa ggagggagat gatcgcccct tcttctggct ctttgagaat gtggtggcca
     4201 tgggcgttag tgacaagagg gacatctcgc gatttcttga gtctaacccc gtgatgattg
     4261 acgccaaaga agtgtctgct gcacacaggg cccgttactt ctggggtaac cttcctggca
     4321 tgaacaggcc tttggcatcc actgtgaatg ataagctgga gctgcaagag tgtctggagc
     4381 acggcagaat agccaagttc agcaaagtga ggaccattac caccaggtca aactctataa
     4441 agcagggcaa agaccagcat ttccccgtct tcatgaacga gaaggaggac atcctgtggt
     4501 gcactgaaat ggaaagggtg tttggcttcc ccgtccacta cacagacgtc tccaacatga
     4561 gccgcttggc gaggcagaga ctgctgggcc gatcgtggag cgtgccggtc atccgccacc
     4621 tcttcgctcc gctgaaggaa tattttgctt gtgtgtctag cggcaatagt aacgctaaca
     4681 gccgcgggcc gagcttcagc agcggcctgg tgccgttaag cttgcgcggc agccatatgg
     4741 gccctatgga gatatacaag acagtgtctg catggaagag acagccagtg cgggtactga
     4801 gcctcttcag aaacatcgac aaggtactaa agagtttggg cttcttggaa agcggttctg
     4861 gttctggggg aggaacgctg aagtacgtgg aagatgtcac aaatgtcgtg aggagagacg
     4921 tggagaaatg gggccccttt gacctggtgt acggctcgac gcagccccta ggcagctctt
     4981 gtgatcgctg tcccggctgg tacatgttcc agttccaccg gatcctgcag tatgcgctgc
     5041 ctcgccagga gagtcagcgg cccttcttct ggatattcat ggacaatctg ctgctgactg
     5101 aggatgacca agagacaact acccgcttcc ttcagacaga ggctgtgacc ctccaggatg
     5161 tccgtggcag agactaccag aatgctatgc gggtgtggag caacattcca gggctgaaga
     5221 gcaagcatgc gcccctgacc ccaaaggaag aagagtatct gcaagcccaa gtcagaagca
     5281 ggagcaagct ggacgccccg aaagttgacc tcctggtgaa gaactgcctt ctcccgctga
     5341 gagagtactt caagtatttt tctcaaaact cacttcctct tggagggccg agctctggcg
     5401 cacccccacc aagtggaggg tctcctgccg ggtccccaac atctactgaa gaaggcacca
     5461 gcgaatccgc aacgcccgag tcaggccctg gtacctccac agaaccatct gaaggtagtg
     5521 cgcctggttc cccagctgga agccctactt ccaccgaaga aggcacgtca accgaaccaa
     5581 gtgaaggatc tgcccctggg accagcactg aaccatctga gatggacaag aagtattcta
     5641 tcggactggc catcgggact aatagcgtcg ggtgggccgt gatcactgac gagtacaagg
     5701 tgccctctaa gaagttcaag gtgctcggga acaccgaccg gcattccatc aagaaaaatc
     5761 tgatcggagc tctcctcttt gattcagggg agaccgctga agcaacccgc ctcaagcgga
     5821 ctgctagacg gcggtacacc aggaggaaga accggatttg ttaccttcaa gagatattct
     5881 ccaacgaaat ggcaaaggtc gacgacagct tcttccatag gctggaagaa tcattcctcg
     5941 tggaagagga taagaagcat gaacggcatc ccatcttcgg taatatcgtc gacgaggtgg
     6001 cctatcacga gaaataccca accatctacc atcttcgcaa aaagctggtg gactcaaccg
     6061 acaaggcaga cctccggctt atctacctgg ccctggccca catgatcaag ttcagaggcc
     6121 acttcctgat cgagggcgac ctcaatcctg acaatagcga tgtggataaa ctgttcatcc
     6181 agctggtgca gacttacaac cagctctttg aagagaaccc catcaatgca agcggagtcg
     6241 atgccaaggc cattctgtca gcccggctgt caaagagccg cagacttgag aatcttatcg
     6301 ctcagctgcc gggtgaaaag aaaaatggac tgttcgggaa cctgattgct ctttcacttg
     6361 ggctgactcc caatttcaag tctaatttcg acctggcaga ggatgccaag ctgcaactgt
     6421 ccaaggacac ctatgatgac gatctcgaca acctcctggc ccagatcggt gaccaatacg
     6481 ccgacctttt ccttgctgct aagaatcttt ctgacgccat cctgctgtct gacattctcc
     6541 gcgtgaacac tgaaatcacc aaggcccctc tttcagcttc aatgattaag cggtatgatg
     6601 agcaccacca ggacctgacc ctgcttaagg cactcgtccg gcagcagctt ccggagaagt
     6661 acaaggaaat cttctttgac cagtcaaaga atggatacgc cggctacatc gacggaggtg
     6721 cctcccaaga ggaattttat aagtttatca aacctatcct tgagaagatg gacggcaccg
     6781 aagagctcct cgtgaaactg aatcgggagg atctgctgcg gaagcagcgc actttcgaca
     6841 atgggagcat tccccaccag atccatcttg gggagcttca cgccatcctt cggcgccaag
     6901 aggacttcta cccctttctt aaggacaaca gggagaagat tgagaaaatt ctcactttcc
     6961 gcatccccta ctacgtggga cccctcgcca gaggaaatag ccggtttgct tggatgacca
     7021 gaaagtcaga agaaactatc actccctgga acttcgaaga ggtggtggac aagggagcca
     7081 gcgctcagtc attcatcgaa cggatgacta acttcgataa gaacctcccc aatgagaagg
     7141 tcctgccgaa acattccctg ctctacgagt actttaccgt gtacaacgag ctgaccaagg
     7201 tgaaatatgt caccgaaggg atgaggaagc ccgcattcct gtcaggcgaa caaaagaagg
     7261 caattgtgga ccttctgttc aagaccaata gaaaggtgac cgtgaagcag ctgaaggagg
     7321 actatttcaa gaaaattgaa tgcttcgact ctgtggagat tagcggggtc gaagatcggt
     7381 tcaacgcaag cctgggtacc taccatgatc tgcttaagat catcaaggac aaggattttc
     7441 tggacaatga ggagaacgag gacatccttg aggacattgt cctgactctc actctgttcg
     7501 aggaccggga aatgatcgag gagaggctta agacctacgc ccatctgttc gacgataaag
     7561 tgatgaagca acttaaacgg agaagatata ccggatgggg acgccttagc cgcaaactca
     7621 tcaacggaat ccgggacaaa cagagcggaa agaccattct tgatttcctt aagagcgacg
     7681 gattcgctaa tcgcaacttc atgcaactta tccatgatga ttccctgacc tttaaggagg
     7741 acatccagaa ggcccaagtg tctggacaag gtgactcact gcacgagcat atcgcaaatc
     7801 tggctggttc acccgctatt aagaagggta ttctccagac cgtgaaagtc gtggacgagc
     7861 tggtcaaggt gatgggtcgc cataaaccag agaacattgt catcgagatg gccagggaaa
     7921 accagactac ccagaaggga cagaagaaca gcagggagcg gatgaaaaga attgaggaag
     7981 ggattaagga gctcgggtca cagatcctta aagagcaccc ggtggaaaac acccagcttc
     8041 agaatgagaa gctctatctg tactaccttc aaaatggacg cgatatgtat gtggaccaag
     8101 agcttgatat caacaggctc tcagactacg acgtggacgc catcgtccct cagagcttcc
     8161 tcaaagacga ctcaattgac aataaggtgc tgactcgctc agacaagaac cggggaaagt
     8221 cagataacgt gccctcagag gaagtcgtga aaaagatgaa gaactattgg cgccagcttc
     8281 tgaacgcaaa gctgatcact cagcggaagt tcgacaatct cactaaggct gagaggggcg
     8341 gactgagcga actggacaaa gcaggattca ttaaacggca acttgtggag actcggcaga
     8401 ttactaaaca tgtcgcccaa atccttgact cacgcatgaa taccaagtac gacgaaaacg
     8461 acaaacttat ccgcgaggtg aaggtgatta ccctgaagtc caagctggtc agcgatttca
     8521 gaaaggactt tcaattctac aaagtgcggg agatcaataa ctatcatcat gctcatgacg
     8581 catatctgaa tgccgtggtg ggaaccgccc tgatcaagaa gtacccaaag ctggaaagcg
     8641 agttcgtgta cggagactac aaggtctacg acgtgcgcaa gatgattgcc aaatctgagc
     8701 aggagatcgg aaaggccacc gcaaagtact tcttctacag caacatcatg aatttcttca
     8761 agaccgaaat cacccttgca aacggtgaga tccggaagag gccgctcatc gagactaatg
     8821 gggagactgg cgaaatcgtg tgggacaagg gcagagattt cgctaccgtg cgcaaagtgc
     8881 tttctatgcc tcaagtgaac atcgtgaaga aaaccgaggt gcaaaccgga ggcttttcta
     8941 aggaatcaat cctccccaag cgcaactccg acaagctcat tgcaaggaag aaggattggg
     9001 accctaagaa gtacggcgga ttcgattcac caactgtggc ttattctgtc ctggtcgtgg
     9061 ctaaggtgga aaaaggaaag tctaagaagc tcaagagcgt gaaggaactg ctgggtatca
     9121 ccattatgga gcgcagctcc ttcgagaaga acccaattga ctttctcgaa gccaaaggtt
     9181 acaaggaagt caagaaggac cttatcatca agctcccaaa gtatagcctg ttcgaactgg
     9241 agaatgggcg gaagcggatg ctcgcctccg ctggcgaact tcagaagggt aatgagctgg
     9301 ctctcccctc caagtacgtg aatttcctct accttgcaag ccattacgag aagctgaagg
     9361 ggagccccga ggacaacgag caaaagcaac tgtttgtgga gcagcataag cattatctgg
     9421 acgagatcat tgagcagatt tccgagtttt ctaaacgcgt cattctcgct gatgccaacc
     9481 tcgataaagt ccttagcgca tacaataagc acagagacaa accaattcgg gagcaggctg
     9541 agaatatcat ccacctgttc accctcacca atcttggtgc ccctgccgca ttcaagtact
     9601 tcgacaccac catcgaccgg aaacgctata cctccaccaa agaagtgctg gacgccaccc
     9661 tcatccacca gagcatcacc ggactttacg aaactcggat tgacctctca cagctcggag
     9721 gggatgccta tccctatgac gtgcccgatt atgccagcct gggcagcggc tcccccaaga
     9781 aaaaacgcaa ggtggaagat cctaagaaaa agcggaaagt ggacggcatt ggtagtggga
     9841 gcaacggcag cagcggatcc agcgagctga ttaaggagaa catgcacatg aagctgtaca
     9901 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
     9961 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
    10021 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
    10081 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
    10141 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
    10201 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
    10261 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
    10321 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca
    10381 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
    10441 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
    10501 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaatggcg
    10561 gtggcggagg gatggatgct aagtcactaa ctgcctggtc ccggacactg gtgaccttca
    10621 aggatgtatt tgtggacttc accagggagg agtggaagct gctggacact gctcagcaga
    10681 tcgtgtacag aaatgtgatg ctggagaact ataagaacct ggtttccttg ggttatcagc
    10741 ttactaagcc agatgtgatc ctccggttgg agaagggaga agagccctag taagcggccg
    10801 cactcgagat atctagaccc agctttcttg tacaaagtgg tgataattct gcagatatcc
    10861 atcacactgg cggccgctcg agcgcccctc tcccctaacg ttactggccg aagccgcttg
    10921 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc
    10981 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc
    11041 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa
    11101 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct
    11161 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca
    11221 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca
    11281 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat
    11341 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc
    11401 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataatc catggccaag
    11461 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
    11521 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
    11581 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
    11641 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
    11701 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
    11761 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
    11821 ctgccctctg gttatgtgtg ggagggctaa gctagcctag tgccatttgt tcagtggttc
    11881 gtagggcttt cccccactgt ttggctttca gttatatgga tgatgtggta ttgggggcca
    11941 agtctgtaca gcatcttgag tcccttttta ccgctgttac caattttctt ttgtctttgg
    12001 gtatacattt aaaccctaac aaaacaaaga gatggggtta ctctctaaat tttatgggtt
    12061 atgtcattgg atgttatggg tccttgccac aagaacacat catacaaaaa atcaaagaat
    12121 gttttagaaa acttcctatt aacaggccta ttgattggaa agtatgtcaa cgaattgtgg
    12181 gtcttttggg ttttgctgcc ccttttacac aatgtggtta tcctgcgttg atgcctttgt
    12241 atgcatgtat tcaatctaag caggctttca ctttctcgcc aacttacaag gcctttctgt
    12301 gtaaacaata cctgaacctt taccccgttg cccggcaacg gccaggtctg tgccaagtgt
    12361 ttgctgacgc aacccccact ggctggggct tggtcatggg ccatcagcgc atgcgtggaa
    12421 ccttttcggc tcctctgccg atccatactg cggaactcct agccgcttgt tttgctcgca
    12481 gcaggtctgg agcaaacatt atcgggactg ataactctgt tgtcctatcc cgcaaatata
    12541 catcgtttcc atggctgcta ggctgtgctg ccaactggat cctgcgcggg acgtcctttg
    12601 tttacgtccc gtcggcgctg aatcctgcgg acgacccttc tcggggtcgc ttgggactct
    12661 ctcgtcccct tctccgtctg ccgttccgac cgaccacggg gcgcacctct ctttacgcgg
    12721 actccccgtc tgtgccttct catctgccgg accgtgtgca cttcgcttca cctctgcacg
    12781 tcgcatggag accaccgtga acgcccacca aatattgccc aaggtcttac ataagaggac
    12841 tcttggactc tcagcaatgt caacgaccga ccttgaggca tacttcaaag actgtttgtt
    12901 taaagactgg gaggagttgg gggaggagat taggttaaag gtctttgtac taggaggctg
    12961 taggcataaa ttggtctgcg caccagcacc atggcgcaat cactagagcg gggtaccttt
    13021 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
    13081 actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc
    13141 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
    13201 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
    13261 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag
    13321 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
    13381 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
    13441 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
    13501 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta
    13561 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
    13621 gaggccgagg ccggatccct tgagtggctt tcatcctgga gcagactttg cagtctgtgg
    13681 actgcaacac aacattgcct ttatgtgtaa ctcttggctg aagctcttac accaatgctg
    13741 ggggacatgt acctcccagg ggcccaggaa gactacggga ggctacacca acgtcaatca
    13801 gaggggcctg tgtagctacc gataagcgga ccctcaagag ggcattagca atagtgttta
    13861 taaggccccc ttgttaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
    13921 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
    13981 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
    14041 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
    14101 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
    14161 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
    14221 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
    14281 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
    14341 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
    14401 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
    14461 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
    14521 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
    14581 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
    14641 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
    14701 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
    14761 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
    14821 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
    14881 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
    14941 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
    15001 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
    15061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
    15121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
    15181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
    15241 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
    15301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
    15361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
    15421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
    15481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
    15541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
    15601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
    15661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
    15721 gcctttttac ggttcctggc cttttgctgg ccttgaagct gtccctgatg gtcgtcatct
    15781 acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga
    15841 atcataatgg ggaaggccat ccagcctcgc gtcg
//
