LOCUS       sequence-373286-       15057 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 15057)
  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 15057)
  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: pJT257 Lenti
            pEF-dCas9-3XFLAG-Z+F-T2A-tagBFP-P...
FEATURES             Location/Qualifiers
     source          1..15057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..51)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        223..602
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        604..802
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     757..777
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             820..1000
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1047..1172
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1665..1898
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2083..2127
                     /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    2425..2542
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2672..3850
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2903..3841
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3798..3818
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3866..3913
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3914..3937,3938..3940,3941..6496,6497..6499,
                     6500..8014)
                     /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             8069..8089
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8105..8170
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8474..8527
                     /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             9254..9310
                     /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             9311..9709
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9734..10322
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9787..9807)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10205..10216)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10325..10341)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10326..10342)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10847..11027
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(11053..11070)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11059..11283
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11329..11757
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11416..11435)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11626..11647
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11766..11786)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11771..12100
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11951..12086
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12013..12032
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12148..12195
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12214..12588
                     /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    12718..12839
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12755..12774)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12809..12828
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12888..12904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12888..12904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12901..12923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12912..12928
                     /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(12936..12942,12943..12960,12961..12966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12981..13002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13119..13136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13290..13878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13370..13389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14049..14840,14841..14909))
                     /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     14672..14691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14910..15014)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
       61 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
      121 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
      181 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
      241 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      301 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      361 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      421 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      481 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      541 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      601 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      661 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      721 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      781 ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag
      841 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      901 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      961 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
     1021 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
     1081 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
     1141 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     1201 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     1261 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     1321 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     1381 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     1441 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     1501 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     1561 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     1621 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     1681 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     1741 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     1801 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     1861 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     1921 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     1981 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     2041 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     2101 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     2161 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     2221 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     2281 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
     2341 gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag
     2401 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     2461 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     2521 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt
     2581 aattagctag ctgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg
     2641 gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg
     2701 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta
     2761 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     2821 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     2881 cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt
     2941 tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct
     3001 tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc
     3061 cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat
     3121 ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt
     3181 ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga
     3241 tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca
     3301 gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta
     3361 gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc
     3421 ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc
     3481 cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga
     3541 gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac
     3601 ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc
     3661 tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac
     3721 tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt
     3781 tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca
     3841 ggtgtcgtga cgtacggcca ccatgaaaag gccggcggcc acgaaaaagg ccggccaggc
     3901 aaaaaagaaa aaggacaaga agtacagcat cggcctggcc atcggcacca actctgtggg
     3961 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     4021 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     4081 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     4141 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     4201 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     4261 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     4321 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     4381 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     4441 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     4501 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     4561 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     4621 gttcggcaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     4681 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     4741 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     4801 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     4861 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     4921 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     4981 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     5041 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     5101 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     5161 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     5221 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     5281 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     5341 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     5401 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     5461 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     5521 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     5581 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     5641 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     5701 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     5761 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     5821 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     5881 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     5941 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     6001 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     6061 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     6121 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     6181 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     6241 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     6301 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     6361 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     6421 tgtggaccac atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     6481 gaccagaagc gacaaggccc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     6541 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     6601 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     6661 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     6721 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     6781 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     6841 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     6901 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     6961 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     7021 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     7081 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     7141 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     7201 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
     7261 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
     7321 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     7381 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     7441 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     7501 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     7561 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     7621 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     7681 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     7741 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     7801 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     7861 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
     7921 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     7981 gacacggatc gacctgtctc agctgggagg cgacagcgct ggaggaggtg gaagcggagg
     8041 aggaggaagc ggaggaggag gtagcggacc taagaaaaag aggaaggtgg cggccgctgg
     8101 atccgactac aaagaccatg acggtgatta taaagatcat gacatcgact acaaggatga
     8161 cgatgacaag ggtggatccg gatcaggctc cttcgtgacc ctgaaggacg tgggcatgga
     8221 cttcaccctg ggcgactggg agcagctggg cctggagcag ggcgacacct tctgggacac
     8281 cgccctggac aactgccagg acctgttcct gctggacccc cccggaggag gaggtagcgg
     8341 acacgagaag ttccccagcg acctggacct ggacatgttc aacggcagcc tggagtgcga
     8401 catggagagc atcatcagaa gcgagctgat ggacgccgac ggcctggact tcaacttcga
     8461 cagcggatct ggagagggaa ggggaagcct cctaacttgc ggagatgtcg aggagaatcc
     8521 tggcccaatg gttagtaagg gagaggagtt gataaaagag aatatgcaca tgaaactgta
     8581 tatggaaggg acagtggaca accatcactt caagtgcacc tctgagggag aaggaaagcc
     8641 atacgaaggc acccagacta tgcgaattaa ggtcgtggag ggagggcctc tccccttcgc
     8701 cttcgatatt cttgcaacat ctttcctcta cgggagcaaa accttcatca atcacacaca
     8761 aggcatacct gactttttta agcagagttt tccagagggc tttacctggg agcgtgtgac
     8821 tacatacgaa gatgggggtg tgctgacggc tacacaggat acttcacttc aggacggttg
     8881 tctgatctac aacgtcaaga tcaggggggt caatttcacc tcgaatggac cagtaatgca
     8941 aaaaaagacc ctgggatggg aagcttttac tgaaactctc tatcccgccg acggcggcct
     9001 cgaaggcaga aacgatatgg ctttgaagct ggttggcggg tcccatctaa ttgccaatgc
     9061 caaaaccaca tataggagca aaaagcctgc aaagaacctg aagatgccgg gtgtgtacta
     9121 tgttgactat agattagagc ggatcaaaga ggcaaataac gaaacgtacg tggagcagca
     9181 cgaggtagcc gtggcgcgct actgcgattt accctccaaa ctgggccata aactgaactg
     9241 tacaggaagc ggagctacta acttcagcct gctgaagcag gctggcgacg tggaagaaaa
     9301 cccaggacct atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     9361 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     9421 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     9481 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     9541 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     9601 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     9661 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag aattcgatat
     9721 caagcttatc ggtaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     9781 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     9841 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     9901 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     9961 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
    10021 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
    10081 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
    10141 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
    10201 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
    10261 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
    10321 gcatcgatac cgtcgacctc gagacctaga aaaacatgga gcaatcacaa gtagcaatac
    10381 agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt
    10441 tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
    10501 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
    10561 tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc agaactacac
    10621 accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
    10681 tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt
    10741 gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg
    10801 cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt
    10861 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
    10921 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
    10981 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa
    11041 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
    11101 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
    11161 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
    11221 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
    11281 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
    11341 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
    11401 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
    11461 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
    11521 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
    11581 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
    11641 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
    11701 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
    11761 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
    11821 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
    11881 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
    11941 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
    12001 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
    12061 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
    12121 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
    12181 atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg
    12241 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
    12301 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
    12361 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
    12421 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
    12481 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
    12541 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga
    12601 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
    12661 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
    12721 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
    12781 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
    12841 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
    12901 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
    12961 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
    13021 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
    13081 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
    13141 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
    13201 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
    13261 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
    13321 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
    13381 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
    13441 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
    13501 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
    13561 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
    13621 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
    13681 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
    13741 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
    13801 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
    13861 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
    13921 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
    13981 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
    14041 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
    14101 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
    14161 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
    14221 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
    14281 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
    14341 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
    14401 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
    14461 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
    14521 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
    14581 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
    14641 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
    14701 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
    14761 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
    14821 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
    14881 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
    14941 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
    15001 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcggga
//
