LOCUS       sequence-373288-       16353 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 16353)
  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 16353)
  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: pJT260 Lenti
            pEF-dCas9-3XFLAG-VPR-T2A-tagBFP-P...
FEATURES             Location/Qualifiers
     source          1..16353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(54..73)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        245..624
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        626..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     779..799
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             842..1022
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1069..1194
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1687..1920
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2105..2149
                     /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    2447..2564
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2694..3872
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2925..3863
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3820..3840
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3888..3935
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3936..3959,3960..3962,3963..6518,6519..6521,
                     6522..8036)
                     /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             8091..8111
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8127..8192
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8214..8363
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             8388..8408
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8838..9194
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             9213..9782
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             9792..9845
                     /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             10572..10628
                     /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             10629..11027
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    11052..11640
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11105..11125)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11523..11534)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11643..11659)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11644..11660)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12165..12345
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(12371..12388)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12377..12601
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12647..13075
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12734..12753)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12944..12965
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(13084..13104)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        13089..13418
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      13269..13404
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     13331..13350
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        13466..13513
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             13532..13906
                     /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    14036..14157
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(14073..14092)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     14127..14146
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(14206..14222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14206..14222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14219..14241)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14230..14246
                     /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(14254..14260,14261..14278,14279..14284))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14299..14320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14437..14454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14608..15196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14688..14707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15367..16158,16159..16227))
                     /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     15990..16009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16228..16332)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
       61 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
      121 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      181 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      241 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      301 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      361 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      421 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      481 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      541 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      601 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      661 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      721 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      781 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt ttgcctgtac
      841 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
      901 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
      961 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     1021 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     1081 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     1141 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     1201 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     1261 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     1321 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     1381 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     1441 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     1501 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     1561 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1621 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1681 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1741 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1801 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1861 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1921 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1981 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     2041 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     2101 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     2161 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     2221 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     2281 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     2341 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     2401 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2461 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2521 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2581 acagcagaga tccagtttgg ttaattagct agctgcaaag atggataaag ttttaaacag
     2641 agaggaatct ttgcagctaa tggaccttct aggtcttgaa aggagtggga attggctccg
     2701 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     2761 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     2821 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     2881 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg
     2941 gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca
     3001 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
     3061 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc
     3121 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
     3181 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata
     3241 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
     3301 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     3361 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     3421 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     3481 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc
     3541 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     3601 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     3661 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     3721 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     3781 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     3841 aaagtttttt tcttccattt caggtgtcgt gacgtacggc caccatgaaa aggccggcgg
     3901 ccacgaaaaa ggccggccag gcaaaaaaga aaaaggacaa gaagtacagc atcggcctgg
     3961 ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     4021 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     4081 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     4141 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     4201 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     4261 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     4321 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     4381 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     4441 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     4501 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     4561 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     4621 ccggcgagaa gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc
     4681 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     4741 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     4801 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     4861 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     4921 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     4981 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     5041 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     5101 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     5161 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     5221 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     5281 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     5341 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     5401 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     5461 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     5521 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     5581 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     5641 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     5701 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     5761 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     5821 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     5881 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     5941 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     6001 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     6061 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     6121 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     6181 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     6241 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     6301 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     6361 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     6421 tcaaccggct gtccgactac gatgtggacc acatcgtgcc tcagagcttt ctgaaggacg
     6481 actccatcga caacaaggtg ctgaccagaa gcgacaaggc ccggggcaag agcgacaacg
     6541 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     6601 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     6661 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     6721 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     6781 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     6841 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     6901 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     6961 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     7021 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     7081 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     7141 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     7201 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     7261 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     7321 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     7381 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     7441 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     7501 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     7561 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     7621 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     7681 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     7741 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     7801 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     7861 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca
     7921 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     7981 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacagcg
     8041 ctggaggagg tggaagcgga ggaggaggaa gcggaggagg aggtagcgga cctaagaaaa
     8101 agaggaaggt ggcggccgct ggatccgact acaaagacca tgacggtgat tataaagatc
     8161 atgacatcga ctacaaggat gacgatgaca agggtggatc cggatcaggc tccgacgcat
     8221 tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt gaccttgaca
     8281 tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt gacgcccttg
     8341 atgatttcga cctggacatg ctgattaact ctagaagttc cggatctccg aaaaagaaac
     8401 gcaaagttgg tagccagtac ctgcccgaca ccgacgaccg gcaccggatc gaggaaaagc
     8461 ggaagcggac ctacgagaca ttcaagagca tcatgaagaa gtcccccttc agcggcccca
     8521 ccgaccctag acctccacct agaagaatcg ccgtgcccag cagatccagc gccagcgtgc
     8581 caaaacctgc cccccagcct taccccttca ccagcagcct gagcaccatc aactacgacg
     8641 agttccctac catggtgttc cccagcggcc agatctctca ggcctctgct ctggctccag
     8701 cccctcctca ggtgctgcct caggctcctg ctcctgcacc agctccagcc atggtgtctg
     8761 cactggctca ggcaccagca cccgtgcctg tgctggctcc tggacctcca caggctgtgg
     8821 ctccaccagc ccctaaacct acacaggccg gcgagggcac actgtctgaa gctctgctgc
     8881 agctgcagtt cgacgacgag gatctgggag ccctgctggg aaacagcacc gatcctgccg
     8941 tgttcaccga cctggccagc gtggacaaca gcgagttcca gcagctgctg aaccagggca
     9001 tccctgtggc ccctcacacc accgagccca tgctgatgga ataccccgag gccatcaccc
     9061 ggctcgtgac aggcgctcag aggcctcctg atccagctcc tgcccctctg ggagcaccag
     9121 gcctgcctaa tggactgctg tctggcgacg aggacttcag ctctatcgcc gatatggatt
     9181 tctcagcctt gctgggctct ggcagcggca gccgggattc cagggaaggg atgtttttgc
     9241 cgaagcctga ggccggctcc gctattagtg acgtgtttga gggccgcgag gtgtgccagc
     9301 caaaacgaat ccggccattt catcctccag gaagtccatg ggccaaccgc ccactccccg
     9361 ccagcctcgc accaacacca accggtccag tacatgagcc agtcgggtca ctgaccccgg
     9421 caccagtccc tcagccactg gatccagcgc ccgcagtgac tcccgaggcc agtcacctgt
     9481 tggaggatcc cgatgaagag acgagccagg ctgtcaaagc ccttcgggag atggccgata
     9541 ctgtgattcc ccagaaggaa gaggctgcaa tctgtggcca aatggacctt tcccatccgc
     9601 ccccaagggg ccatctggat gagctgacaa ccacacttga gtccatgacc gaggatctga
     9661 acctggactc acccctgacc ccggaattga acgagattct ggataccttc ctgaacgacg
     9721 agtgcctctt gcatgccatg catatcagca caggactgtc catcttcgac acatctctgt
     9781 ttggatctgg agagggaagg ggaagcctcc taacttgcgg agatgtcgag gagaatcctg
     9841 gcccaatggt tagtaaggga gaggagttga taaaagagaa tatgcacatg aaactgtata
     9901 tggaagggac agtggacaac catcacttca agtgcacctc tgagggagaa ggaaagccat
     9961 acgaaggcac ccagactatg cgaattaagg tcgtggaggg agggcctctc cccttcgcct
    10021 tcgatattct tgcaacatct ttcctctacg ggagcaaaac cttcatcaat cacacacaag
    10081 gcatacctga cttttttaag cagagttttc cagagggctt tacctgggag cgtgtgacta
    10141 catacgaaga tgggggtgtg ctgacggcta cacaggatac ttcacttcag gacggttgtc
    10201 tgatctacaa cgtcaagatc aggggggtca atttcacctc gaatggacca gtaatgcaaa
    10261 aaaagaccct gggatgggaa gcttttactg aaactctcta tcccgccgac ggcggcctcg
    10321 aaggcagaaa cgatatggct ttgaagctgg ttggcgggtc ccatctaatt gccaatgcca
    10381 aaaccacata taggagcaaa aagcctgcaa agaacctgaa gatgccgggt gtgtactatg
    10441 ttgactatag attagagcgg atcaaagagg caaataacga aacgtacgtg gagcagcacg
    10501 aggtagccgt ggcgcgctac tgcgatttac cctccaaact gggccataaa ctgaactgta
    10561 caggaagcgg agctactaac ttcagcctgc tgaagcaggc tggcgacgtg gaagaaaacc
    10621 caggacctat ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg
    10681 ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta
    10741 gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag
    10801 aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg
    10861 cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc
    10921 tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag
    10981 ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaagaa ttcgatatca
    11041 agcttatcgg taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
    11101 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
    11161 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
    11221 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
    11281 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
    11341 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
    11401 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
    11461 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
    11521 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
    11581 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
    11641 atcgataccg tcgacctcga gacctagaaa aacatggagc aatcacaagt agcaatacag
    11701 cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc
    11761 cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
    11821 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata
    11881 tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag aactacacac
    11941 cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg
    12001 agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga
    12061 gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc
    12121 tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag
    12181 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
    12241 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
    12301 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
    12361 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
    12421 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
    12481 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
    12541 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
    12601 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
    12661 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
    12721 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
    12781 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
    12841 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
    12901 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
    12961 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
    13021 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
    13081 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
    13141 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
    13201 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
    13261 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
    13321 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
    13381 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
    13441 ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat
    13501 agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt
    13561 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
    13621 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
    13681 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
    13741 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
    13801 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
    13861 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga
    13921 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
    13981 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
    14041 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
    14101 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
    14161 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
    14221 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
    14281 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
    14341 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
    14401 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
    14461 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
    14521 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
    14581 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
    14641 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
    14701 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
    14761 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
    14821 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
    14881 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
    14941 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
    15001 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
    15061 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
    15121 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
    15181 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
    15241 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
    15301 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
    15361 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
    15421 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
    15481 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
    15541 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
    15601 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
    15661 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
    15721 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
    15781 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
    15841 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
    15901 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
    15961 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
    16021 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
    16081 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
    16141 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
    16201 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
    16261 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
    16321 aggggttccg cgcacatttc cccgaaaagt gcc
//
