LOCUS       sequence-369753-       15024 bp    DNA     circular SYN 15-APR-2025
DEFINITION  dCas9 effector fusion for manipulating transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15024)
  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 15024)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pJT351 Lenti
            pEF-dCas9-3XFLAG-P66_CC-T2A-tagBF...
FEATURES             Location/Qualifiers
     source          1..15024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..71)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        243..622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        624..822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     777..797
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             840..1020
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1067..1192
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1685..1918
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2103..2147
                     /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    2445..2562
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2692..3870
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2923..3861
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3818..3838
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3886..3933
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3934..3957,3958..3960,3961..6516,6517..6519,
                     6520..8034)
                     /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             8089..8109
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8125..8190
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8461..8514
                     /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             9241..9297
                     /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             9298..9696
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9721..10309
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9774..9794)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10192..10203)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10312..10328)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10313..10329)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10834..11014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(11040..11057)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11046..11270
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11316..11744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11403..11422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11613..11634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11753..11773)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11758..12087
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11938..12073
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12000..12019
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12135..12182
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12201..12575
                     /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    12705..12826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12742..12761)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12796..12815
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12875..12891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12875..12891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12888..12910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12899..12915
                     /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(12923..12929,12930..12947,12948..12953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12968..12989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13106..13123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13277..13865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13357..13376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14036..14827,14828..14896))
                     /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     14659..14678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14897..15001)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
       61 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
      121 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      181 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      241 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      301 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      361 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      421 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      481 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      541 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      601 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      661 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      721 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      781 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg
      841 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
      901 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
      961 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     1021 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     1081 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     1141 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     1201 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     1261 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     1321 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     1381 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     1441 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     1501 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
     1561 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     1621 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     1681 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     1741 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     1801 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     1861 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     1921 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     1981 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     2041 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     2101 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     2161 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     2221 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     2281 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     2341 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     2401 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     2461 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2521 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     2581 agcagagatc cagtttggtt aattagctag ctgcaaagat ggataaagtt ttaaacagag
     2641 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
     2701 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2761 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2821 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2881 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
     2941 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
     3001 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
     3061 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
     3121 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     3181 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     3241 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     3301 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     3361 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     3421 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     3481 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     3541 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     3601 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     3661 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     3721 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     3781 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     3841 agtttttttc ttccatttca ggtgtcgtga cgtacggcca ccatgaaaag gccggcggcc
     3901 acgaaaaagg ccggccaggc aaaaaagaaa aaggacaaga agtacagcat cggcctggcc
     3961 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     4021 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     4081 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     4141 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     4201 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     4261 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     4321 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     4381 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     4441 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     4501 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     4561 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     4621 ggcgagaaga agaatggcct gttcggcaac ctgattgccc tgagcctggg cctgaccccc
     4681 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     4741 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     4801 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     4861 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     4921 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     4981 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     5041 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     5101 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     5161 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     5221 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     5281 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     5341 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     5401 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     5461 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     5521 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     5581 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     5641 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     5701 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     5761 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     5821 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     5881 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     5941 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     6001 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     6061 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     6121 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     6181 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     6241 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     6301 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     6361 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     6421 aaccggctgt ccgactacga tgtggaccac atcgtgcctc agagctttct gaaggacgac
     6481 tccatcgaca acaaggtgct gaccagaagc gacaaggccc ggggcaagag cgacaacgtg
     6541 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     6601 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     6661 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     6721 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     6781 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     6841 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     6901 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     6961 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     7021 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     7081 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     7141 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     7201 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     7261 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     7321 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     7381 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     7441 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     7501 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     7561 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     7621 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     7681 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     7741 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     7801 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     7861 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     7921 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
     7981 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacagcgct
     8041 ggaggaggtg gaagcggagg aggaggaagc ggaggaggag gtagcggacc taagaaaaag
     8101 aggaaggtgg cggccgctgg atccgactac aaagaccatg acggtgatta taaagatcat
     8161 gacatcgact acaaggatga cgatgacaag ggtggatccg gatcaggctc caacggcctg
     8221 accaccgtgg ccctgaagga gaccagcacc gaggccctga tgaagagcag ccccgaggag
     8281 agagagagaa tgatcaagca gctgaaggag gagctgagac tggaggaggc caagctggtg
     8341 ctgctgaaga agctgagaca gagccagatc cagaaggagg ccaccgccca gaagcccacc
     8401 ggcagcgtgg gcagcaccgt gaccacaccc cctcccctgg tgagaggcac cggatctgga
     8461 gagggaaggg gaagcctcct aacttgcgga gatgtcgagg agaatcctgg cccaatggtt
     8521 agtaagggag aggagttgat aaaagagaat atgcacatga aactgtatat ggaagggaca
     8581 gtggacaacc atcacttcaa gtgcacctct gagggagaag gaaagccata cgaaggcacc
     8641 cagactatgc gaattaaggt cgtggaggga gggcctctcc ccttcgcctt cgatattctt
     8701 gcaacatctt tcctctacgg gagcaaaacc ttcatcaatc acacacaagg catacctgac
     8761 ttttttaagc agagttttcc agagggcttt acctgggagc gtgtgactac atacgaagat
     8821 gggggtgtgc tgacggctac acaggatact tcacttcagg acggttgtct gatctacaac
     8881 gtcaagatca ggggggtcaa tttcacctcg aatggaccag taatgcaaaa aaagaccctg
     8941 ggatgggaag cttttactga aactctctat cccgccgacg gcggcctcga aggcagaaac
     9001 gatatggctt tgaagctggt tggcgggtcc catctaattg ccaatgccaa aaccacatat
     9061 aggagcaaaa agcctgcaaa gaacctgaag atgccgggtg tgtactatgt tgactataga
     9121 ttagagcgga tcaaagaggc aaataacgaa acgtacgtgg agcagcacga ggtagccgtg
     9181 gcgcgctact gcgatttacc ctccaaactg ggccataaac tgaactgtac aggaagcgga
     9241 gctactaact tcagcctgct gaagcaggct ggcgacgtgg aagaaaaccc aggacctatg
     9301 gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac
     9361 agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc
     9421 atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg
     9481 ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat
     9541 gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat
     9601 cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt
     9661 gaattgctgc cctctggtta tgtgtgggag ggctaagaat tcgatatcaa gcttatcggt
     9721 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     9781 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     9841 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     9901 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     9961 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
    10021 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
    10081 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
    10141 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
    10201 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
    10261 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
    10321 cgacctcgag acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat
    10381 gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct
    10441 caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
    10501 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg
    10561 tggatctacc acacacaagg ctacttccct gattggcaga actacacacc agggccaggg
    10621 atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag
    10681 gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg
    10741 atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat
    10801 cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga
    10861 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
    10921 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
    10981 agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc gctgatcagc
    11041 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
    11101 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
    11161 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
    11221 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
    11281 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
    11341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
    11401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
    11461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
    11521 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
    11581 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
    11641 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
    11701 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
    11761 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
    11821 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
    11881 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
    11941 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
    12001 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
    12061 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
    12121 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
    12181 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
    12241 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
    12301 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
    12361 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
    12421 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
    12481 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
    12541 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
    12601 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
    12661 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
    12721 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
    12781 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
    12841 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
    12901 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
    12961 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
    13021 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
    13081 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
    13141 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
    13201 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
    13261 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
    13321 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
    13381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
    13441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
    13501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
    13561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
    13621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
    13681 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
    13741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
    13801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
    13861 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
    13921 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
    13981 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
    14041 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
    14101 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
    14161 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
    14221 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
    14281 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
    14341 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
    14401 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
    14461 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
    14521 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
    14581 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
    14641 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
    14701 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
    14761 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
    14821 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
    14881 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
    14941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
    15001 gcacatttcc ccgaaaagtg ccac
//
