LOCUS       sequence-387188-       15033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 effector fusion for manipulating transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15033)
  AUTHORS   Tycko J, Van MV, Aradhana, DelRosso N, Ye H, Yao D, Valbuena R, 
            Vaughan-Jackson A, Xu X, Ludwig C, Spees K, Liu K, Gu M, Khare V, 
            Mukund AX, Suzuki PH, Arana S, Zhang C, Du PP, Ornstein TS, Hess GT,
            Kamber RA, Qi LS, Khalil AS, Bintu L, Bassik MC
  TITLE     Development of compact transcriptional effectors using 
            high-throughput measurements in diverse contexts.
  JOURNAL   Nat Biotechnol. 2024 Nov 1. doi: 10.1038/s41587-024-02442-6.
  PUBMED    39487265
REFERENCE   2  (bases 1 to 15033)
  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: "Nat 
            Biotechnol. 2024 Nov 1. doi: 10.1038/s41587-024-02442-6."
COMMENT     Sequence Label: pJT342 Lenti pEF-dCas9-3XFLAG-RYBP +
            MGA1+2-T2...
FEATURES             Location/Qualifiers
     source          1..15033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        260..458
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     413..433
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             476..656
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    703..828
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1321..1554
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1739..1783
                     /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    2081..2198
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2328..3506
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2559..3497
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3454..3474
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3522..3569
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3570..3593,3594..3596,3597..6152,6153..6155,
                     6156..7670)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7725..7745
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7761..7826
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8106..8159
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             8886..8942
                     /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             8943..9341
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9366..9954
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9419..9439)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9837..9848)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9957..9973)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9958..9974)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10479..10659
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10685..10702)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10691..10915
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10961..11389
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11048..11067)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11258..11279
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11398..11418)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11403..11732
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11583..11718
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11645..11664
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11780..11827
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11846..12220
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    12350..12471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12387..12406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12441..12460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12520..12536)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12520..12536)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12533..12555)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12544..12560
                     /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)."
     promoter        complement(join(12568..12574,12575..12592,12593..12598))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12613..12634
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12751..12768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12922..13510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13002..13021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13681..14472,14473..14541))
                     /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     14304..14323
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14542..14646)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(14721..14740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(14912..15033,1..258)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
       61 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      121 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      181 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      241 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      301 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      361 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      421 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
      481 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      541 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      601 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      661 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      721 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      781 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      841 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      901 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
      961 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1021 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1081 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1141 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1201 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1261 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1321 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1381 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1441 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1501 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1561 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1621 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     1681 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     1741 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     1801 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     1861 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     1921 atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga
     1981 agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg
     2041 tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     2101 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2161 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2221 gagatccagt ttggttaatt agctagctgc aaagatggat aaagttttaa acagagagga
     2281 atctttgcag ctaatggacc ttctaggtct tgaaaggagt gggaattggc tccggtgccc
     2341 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     2401 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     2461 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     2521 acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc
     2581 gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc
     2641 tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc
     2701 cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg
     2761 gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc
     2821 tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg
     2881 taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg
     2941 gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga
     3001 gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc
     3061 cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg
     3121 aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg
     3181 gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg
     3241 cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct
     3301 tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca
     3361 ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat
     3421 ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt
     3481 tttttcttcc atttcaggtg tcgtgacgta cggccaccat gaaaaggccg gcggccacga
     3541 aaaaggccgg ccaggcaaaa aagaaaaagg acaagaagta cagcatcggc ctggccatcg
     3601 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     3661 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     3721 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     3781 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     3841 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     3901 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     3961 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     4021 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     4081 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     4141 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     4201 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     4261 agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact
     4321 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     4381 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     4441 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     4501 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     4561 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     4621 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     4681 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     4741 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     4801 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     4861 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     4921 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     4981 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     5041 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     5101 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     5161 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     5221 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     5281 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     5341 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     5401 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     5461 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     5521 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     5581 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     5641 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     5701 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     5761 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     5821 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     5881 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     5941 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     6001 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     6061 ggctgtccga ctacgatgtg gaccacatcg tgcctcagag ctttctgaag gacgactcca
     6121 tcgacaacaa ggtgctgacc agaagcgaca aggcccgggg caagagcgac aacgtgccct
     6181 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     6241 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     6301 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     6361 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     6421 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     6481 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     6541 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     6601 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     6661 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     6721 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     6781 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     6841 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     6901 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     6961 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     7021 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     7081 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     7141 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     7201 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     7261 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     7321 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     7381 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     7441 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     7501 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     7561 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     7621 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac agcgctggag
     7681 gaggtggaag cggaggagga ggaagcggag gaggaggtag cggacctaag aaaaagagga
     7741 aggtggcggc cgctggatcc gactacaaag accatgacgg tgattataaa gatcatgaca
     7801 tcgactacaa ggatgacgat gacaagggtg gatccggatc aggctcccct aggttgaaga
     7861 atgttgatag atccaccgct caacagttgg ccgtgactgt gggaaatgtc acagtcatta
     7921 tcacagattt taaggaaaaa accggaggag gaggtagcgg aatcaaggag gagcccctgg
     7981 acgactacga cggcagctca tcttcccccg aaaatgatga tctctttatg atgccacgga
     8041 ttgtaaatgt tacatccctc gctacggaag gcggattggt cgatatgggt ggttctggat
     8101 ctggagaggg aaggggaagc ctcctaactt gcggagatgt cgaggagaat cctggcccaa
     8161 tggttagtaa gggagaggag ttgataaaag agaatatgca catgaaactg tatatggaag
     8221 ggacagtgga caaccatcac ttcaagtgca cctctgaggg agaaggaaag ccatacgaag
     8281 gcacccagac tatgcgaatt aaggtcgtgg agggagggcc tctccccttc gccttcgata
     8341 ttcttgcaac atctttcctc tacgggagca aaaccttcat caatcacaca caaggcatac
     8401 ctgacttttt taagcagagt tttccagagg gctttacctg ggagcgtgtg actacatacg
     8461 aagatggggg tgtgctgacg gctacacagg atacttcact tcaggacggt tgtctgatct
     8521 acaacgtcaa gatcaggggg gtcaatttca cctcgaatgg accagtaatg caaaaaaaga
     8581 ccctgggatg ggaagctttt actgaaactc tctatcccgc cgacggcggc ctcgaaggca
     8641 gaaacgatat ggctttgaag ctggttggcg ggtcccatct aattgccaat gccaaaacca
     8701 catataggag caaaaagcct gcaaagaacc tgaagatgcc gggtgtgtac tatgttgact
     8761 atagattaga gcggatcaaa gaggcaaata acgaaacgta cgtggagcag cacgaggtag
     8821 ccgtggcgcg ctactgcgat ttaccctcca aactgggcca taaactgaac tgtacaggaa
     8881 gcggagctac taacttcagc ctgctgaagc aggctggcga cgtggaagaa aacccaggac
     8941 ctatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa
     9001 tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
     9061 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg
     9121 tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
     9181 gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc
     9241 tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga
     9301 ttcgtgaatt gctgccctct ggttatgtgt gggagggcta agaattcgat atcaagctta
     9361 tcggtaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     9421 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     9481 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     9541 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     9601 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     9661 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     9721 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     9781 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     9841 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     9901 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     9961 accgtcgacc tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta
    10021 ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca
    10081 cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
    10141 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg
    10201 atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc
    10261 cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag
    10321 agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc
    10381 atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat
    10441 ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga
    10501 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
    10561 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
    10621 ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga
    10681 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
    10741 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
    10801 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
    10861 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
    10921 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
    10981 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
    11041 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
    11101 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
    11161 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
    11221 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
    11281 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
    11341 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
    11401 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
    11461 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
    11521 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
    11581 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
    11641 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
    11701 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
    11761 tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata
    11821 atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac
    11881 cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga
    11941 cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc
    12001 ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga
    12061 cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc
    12121 ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc
    12181 cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga
    12241 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
    12301 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
    12361 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
    12421 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
    12481 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
    12541 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
    12601 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
    12661 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
    12721 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
    12781 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
    12841 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
    12901 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
    12961 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
    13021 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
    13081 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
    13141 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
    13201 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
    13261 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
    13321 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
    13381 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
    13441 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
    13501 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
    13561 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
    13621 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
    13681 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
    13741 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
    13801 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
    13861 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
    13921 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
    13981 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
    14041 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
    14101 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
    14161 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
    14221 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
    14281 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
    14341 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
    14401 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
    14461 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
    14521 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
    14581 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
    14641 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
    14701 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
    14761 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
    14821 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
    14881 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
    14941 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
    15001 acggtaaatg gcccgcctgg ctgaccgccc aac
//
