LOCUS       sequence-387213-       15348 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 15348)
  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 15348)
  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: pJT331 Lenti
            pEF-dCas9-3XFLAG-Q+N+Z+F-T2A-tagB...
FEATURES             Location/Qualifiers
     source          1..15348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2687..3865
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2918..3856
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3813..3833
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3881..3928
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3929..3952,3953..3955,3956..6511,6512..6514,
                     6515..8029)
                     /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             8084..8104
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8120..8185
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8780..8833
                     /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             9560..9616
                     /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             9617..10015
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    10040..10628
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10093..10113)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10511..10522)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10631..10647)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10632..10648)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11153..11333
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(11359..11376)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11365..11589
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11635..12063
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11722..11741)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11932..11953
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12072..12092)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12077..12406
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      12257..12392
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12319..12338
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12454..12501
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12520..12894
                     /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    13024..13145
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13061..13080)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13115..13134
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13194..13210)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13194..13210)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13207..13229)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13218..13234
                     /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(13242..13248,13249..13266,13267..13272))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13287..13308
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13425..13442)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13596..14184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13676..13695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14355..15146,15147..15215))
                     /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     14978..14997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15216..15320)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta gctagctgca aagatggata aagttttaaa cagagaggaa
     2641 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg
     2701 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     2761 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     2821 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     2881 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
     2941 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
     3001 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
     3061 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
     3121 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
     3181 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
     3241 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
     3301 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
     3361 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
     3421 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
     3481 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     3541 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     3601 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
     3661 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     3721 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     3781 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     3841 ttttcttcca tttcaggtgt cgtgacgtac ggccaccatg aaaaggccgg cggccacgaa
     3901 aaaggccggc caggcaaaaa agaaaaagga caagaagtac agcatcggcc tggccatcgg
     3961 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     4021 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     4081 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     4141 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     4201 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     4261 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     4321 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     4381 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     4441 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     4501 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     4561 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     4621 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
     4681 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     4741 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     4801 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     4861 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     4921 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     4981 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     5041 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     5101 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     5161 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     5221 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     5281 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     5341 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     5401 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     5461 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     5521 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     5581 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     5641 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     5701 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     5761 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     5821 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     5881 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     5941 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     6001 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     6061 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     6121 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     6181 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     6241 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     6301 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     6361 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     6421 gctgtccgac tacgatgtgg accacatcgt gcctcagagc tttctgaagg acgactccat
     6481 cgacaacaag gtgctgacca gaagcgacaa ggcccggggc aagagcgaca acgtgccctc
     6541 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     6601 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     6661 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     6721 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     6781 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     6841 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     6901 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     6961 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     7021 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     7081 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     7141 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     7201 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     7261 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     7321 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     7381 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     7441 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     7501 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     7561 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     7621 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     7681 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     7741 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     7801 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     7861 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     7921 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     7981 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca gcgctggagg
     8041 aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa
     8101 ggtggcggcc gctggatccg actacaaaga ccatgacggt gattataaag atcatgacat
     8161 cgactacaag gatgacgatg acaagggtgg atccggatca ggctccagtc cgttttcccc
     8221 tgttcaactc catcaacttc gggcgcaaat tcttgcatat aaaatgctcg ctcgtggtca
     8281 accgttgcca gaaactcttc aacttgcagt ccagggtggc ggtggtggct ccggcgaagg
     8341 tcaatccgat gaacgcgcac ttcttgatca acttcataca ttgcttagta atacagatgc
     8401 aactggtctc gaagaaattg atcgggcgct cgggatacct gaacttgtca atcaaggtca
     8461 agcacttgaa cctaaacaag gcggcggtgg atcaggtttt gttacattga aagatgtagg
     8521 gatggatttt acactcgggg attgggaaca attggggctc gaacaagggg atacgttttg
     8581 ggatactgct ctcgataatt gtcaagatct ctttctcctc gatccacctg gcggcggtgg
     8641 gtctggccat gagaaatttc cgtctgatct cgatttggat atgtttaatg ggtcactcga
     8701 atgtgatatg gaatcaatta ttcgcagtga actcatggat gcagatggac tcgattttaa
     8761 ttttgattca ggatctggag agggaagggg aagcctccta acttgcggag atgtcgagga
     8821 gaatcctggc ccaatggtta gtaagggaga ggagttgata aaagagaata tgcacatgaa
     8881 actgtatatg gaagggacag tggacaacca tcacttcaag tgcacctctg agggagaagg
     8941 aaagccatac gaaggcaccc agactatgcg aattaaggtc gtggagggag ggcctctccc
     9001 cttcgccttc gatattcttg caacatcttt cctctacggg agcaaaacct tcatcaatca
     9061 cacacaaggc atacctgact tttttaagca gagttttcca gagggcttta cctgggagcg
     9121 tgtgactaca tacgaagatg ggggtgtgct gacggctaca caggatactt cacttcagga
     9181 cggttgtctg atctacaacg tcaagatcag gggggtcaat ttcacctcga atggaccagt
     9241 aatgcaaaaa aagaccctgg gatgggaagc ttttactgaa actctctatc ccgccgacgg
     9301 cggcctcgaa ggcagaaacg atatggcttt gaagctggtt ggcgggtccc atctaattgc
     9361 caatgccaaa accacatata ggagcaaaaa gcctgcaaag aacctgaaga tgccgggtgt
     9421 gtactatgtt gactatagat tagagcggat caaagaggca aataacgaaa cgtacgtgga
     9481 gcagcacgag gtagccgtgg cgcgctactg cgatttaccc tccaaactgg gccataaact
     9541 gaactgtaca ggaagcggag ctactaactt cagcctgctg aagcaggctg gcgacgtgga
     9601 agaaaaccca ggacctatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag
     9661 agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc
     9721 tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc
     9781 ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg
     9841 tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca
     9901 ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc
     9961 gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctaagaatt
    10021 cgatatcaag cttatcggta atcaacctct ggattacaaa atttgtgaaa gattgactgg
    10081 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
    10141 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
    10201 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
    10261 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
    10321 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
    10381 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
    10441 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
    10501 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
    10561 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
    10621 ctccccgcat cgataccgtc gacctcgaga cctagaaaaa catggagcaa tcacaagtag
    10681 caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt
    10741 gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga
    10801 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
    10861 acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa
    10921 ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct acaagctagt
    10981 accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc gcttgttaca
    11041 ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt ggaggtttga
    11101 cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta ctgggtctct
    11161 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
    11221 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
    11281 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagggcccg
    11341 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
    11401 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
    11461 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
    11521 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
    11581 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
    11641 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
    11701 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
    11761 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
    11821 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
    11881 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
    11941 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
    12001 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
    12061 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
    12121 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
    12181 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
    12241 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
    12301 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
    12361 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
    12421 ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat
    12481 atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc
    12541 gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc
    12601 gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc
    12661 ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg
    12721 gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg
    12781 gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc
    12841 ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg
    12901 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
    12961 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
    13021 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
    13081 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
    13141 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
    13201 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
    13261 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
    13321 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
    13381 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
    13441 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
    13501 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
    13561 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
    13621 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
    13681 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
    13741 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
    13801 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
    13861 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
    13921 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
    13981 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
    14041 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
    14101 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
    14161 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
    14221 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
    14281 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
    14341 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
    14401 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
    14461 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
    14521 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
    14581 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
    14641 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
    14701 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
    14761 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
    14821 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
    14881 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
    14941 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
    15001 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
    15061 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
    15121 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
    15181 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
    15241 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
    15301 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgac
//
