LOCUS       sequence-369787-       14916 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 14916)
  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 14916)
  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: pJT252 Lenti
            pEF-dCas9-3XFLAG-ZN473_TAD-T2A-ta...
FEATURES             Location/Qualifiers
     source          1..14916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..50)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        222..601
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        603..801
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     756..776
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             819..999
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1046..1171
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1664..1897
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2082..2126
                     /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    2424..2541
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2671..3849
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2902..3840
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3797..3817
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3865..3912
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3913..3936,3937..3939,3940..6495,6496..6498,
                     6499..8013)
                     /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             8068..8088
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8104..8169
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8332..8385
                     /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             9112..9168
                     /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             9169..9567
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9592..10180
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9645..9665)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10063..10074)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10183..10199)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10184..10200)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10705..10885
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10911..10928)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10917..11141
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11187..11615
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11274..11293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11484..11505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11624..11644)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11629..11958
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11809..11944
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11871..11890
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12006..12053
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12072..12446
                     /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    12576..12697
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12613..12632)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12667..12686
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12746..12762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12746..12762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12759..12781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12770..12786
                     /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(12794..12800,12801..12818,12819..12824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12839..12860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12977..12994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13148..13736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13228..13247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13907..14698,14699..14767))
                     /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     14530..14549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14768..14872)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
       61 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
      121 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
      181 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      241 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      301 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      361 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      421 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      481 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      541 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      601 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      661 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      721 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      781 gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg ttagaccaga
      841 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      901 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
      961 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     1021 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     1081 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     1141 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     1201 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     1261 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     1321 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1381 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     1441 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1501 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     1561 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     1621 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     1681 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     1741 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     1801 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     1861 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     1921 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     1981 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     2041 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     2101 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     2161 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     2221 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     2281 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
     2341 acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga
     2401 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     2461 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     2521 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta
     2581 attagctagc tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg
     2641 accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc
     2701 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag
     2761 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     2821 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     2881 gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt
     2941 acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt
     3001 gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc
     3061 ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc
     3121 tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt
     3181 tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat
     3241 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag
     3301 cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag
     3361 tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc
     3421 tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc
     3481 ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag
     3541 tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg
     3601 gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct
     3661 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact
     3721 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt
     3781 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag
     3841 gtgtcgtgac gtacggccac catgaaaagg ccggcggcca cgaaaaaggc cggccaggca
     3901 aaaaagaaaa aggacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc
     3961 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     4021 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     4081 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     4141 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     4201 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     4261 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     4321 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     4381 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     4441 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     4501 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     4561 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     4621 ttcggcaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     4681 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     4741 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     4801 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     4861 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     4921 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     4981 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     5041 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     5101 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     5161 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     5221 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     5281 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     5341 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     5401 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     5461 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     5521 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     5581 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     5641 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     5701 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     5761 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     5821 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     5881 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     5941 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     6001 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     6061 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     6121 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     6181 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     6241 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     6301 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     6361 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     6421 gtggaccaca tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     6481 accagaagcg acaaggcccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     6541 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     6601 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     6661 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     6721 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     6781 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     6841 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     6901 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     6961 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     7021 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     7081 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     7141 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     7201 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     7261 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     7321 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     7381 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     7441 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     7501 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     7561 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     7621 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     7681 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     7741 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     7801 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     7861 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     7921 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     7981 acacggatcg acctgtctca gctgggaggc gacagcgctg gaggaggtgg aagcggagga
     8041 ggaggaagcg gaggaggagg tagcggacct aagaaaaaga ggaaggtggc ggccgctgga
     8101 tccgactaca aagaccatga cggtgattat aaagatcatg acatcgacta caaggatgac
     8161 gatgacaagg gtggatccgg atcaggctcc ttcgtgaccc tgaaggacgt gggcatggac
     8221 ttcaccctgg gcgactggga gcagctgggc ctggagcagg gcgacacctt ctgggacacc
     8281 gccctggaca actgccagga cctgttcctg ctggaccccc ccggatctgg agagggaagg
     8341 ggaagcctcc taacttgcgg agatgtcgag gagaatcctg gcccaatggt tagtaaggga
     8401 gaggagttga taaaagagaa tatgcacatg aaactgtata tggaagggac agtggacaac
     8461 catcacttca agtgcacctc tgagggagaa ggaaagccat acgaaggcac ccagactatg
     8521 cgaattaagg tcgtggaggg agggcctctc cccttcgcct tcgatattct tgcaacatct
     8581 ttcctctacg ggagcaaaac cttcatcaat cacacacaag gcatacctga cttttttaag
     8641 cagagttttc cagagggctt tacctgggag cgtgtgacta catacgaaga tgggggtgtg
     8701 ctgacggcta cacaggatac ttcacttcag gacggttgtc tgatctacaa cgtcaagatc
     8761 aggggggtca atttcacctc gaatggacca gtaatgcaaa aaaagaccct gggatgggaa
     8821 gcttttactg aaactctcta tcccgccgac ggcggcctcg aaggcagaaa cgatatggct
     8881 ttgaagctgg ttggcgggtc ccatctaatt gccaatgcca aaaccacata taggagcaaa
     8941 aagcctgcaa agaacctgaa gatgccgggt gtgtactatg ttgactatag attagagcgg
     9001 atcaaagagg caaataacga aacgtacgtg gagcagcacg aggtagccgt ggcgcgctac
     9061 tgcgatttac cctccaaact gggccataaa ctgaactgta caggaagcgg agctactaac
     9121 ttcagcctgc tgaagcaggc tggcgacgtg gaagaaaacc caggacctat ggccaagcct
     9181 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc
     9241 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact
     9301 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact
     9361 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg
     9421 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc
     9481 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
     9541 ccctctggtt atgtgtggga gggctaagaa ttcgatatca agcttatcgg taatcaacct
     9601 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     9661 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     9721 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     9781 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     9841 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     9901 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     9961 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
    10021 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
    10081 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
    10141 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga
    10201 gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt
    10261 gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct
    10321 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
    10381 ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac
    10441 cacacacaag gctacttccc tgattggcag aactacacac cagggccagg gatcagatat
    10501 ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa
    10561 gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac
    10621 ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca tcacatggcc
    10681 cgagagctgc atccggactg tactgggtct ctctggttag accagatctg agcctgggag
    10741 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
    10801 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
    10861 tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag cctcgactgt
    10921 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
    10981 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
    11041 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
    11101 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
    11161 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
    11221 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
    11281 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
    11341 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
    11401 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
    11461 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
    11521 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
    11581 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
    11641 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
    11701 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
    11761 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
    11821 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
    11881 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
    11941 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag
    12001 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
    12061 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
    12121 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
    12181 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
    12241 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
    12301 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
    12361 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
    12421 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct
    12481 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
    12541 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
    12601 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
    12661 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
    12721 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
    12781 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
    12841 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
    12901 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
    12961 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
    13021 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
    13081 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
    13141 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
    13201 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
    13261 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
    13321 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
    13381 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
    13441 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
    13501 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
    13561 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
    13621 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
    13681 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
    13741 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
    13801 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
    13861 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
    13921 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
    13981 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
    14041 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
    14101 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
    14161 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
    14221 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
    14281 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
    14341 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
    14401 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
    14461 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
    14521 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
    14581 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
    14641 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
    14701 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
    14761 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
    14821 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
    14881 cccgaaaagt gccacctgac gtcgacggat cgggag
//
