LOCUS       sequence-211560-       10319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-regulated miR-E (miR-30 variant)-based RNAi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10319)
  AUTHORS   Fellmann C, Hoffmann T, Sridhar V, Hopfgartner B, Muhar M, Roth M, 
            Lai DY, Barbosa IA, Kwon JS, Guan Y, Sinha N, Zuber J
  TITLE     An optimized microRNA backbone for effective single-copy RNAi.
  JOURNAL   Cell Rep. 2013 Dec 26;5(6):1704-13. doi: 
            10.1016/j.celrep.2013.11.020. Epub 2013 Dec 12.
  PUBMED    24332856
REFERENCE   2  (bases 1 to 10319)
  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"; doi: 
            "10.1016/j.celrep.2013.11.020"; journalName: "Cell Rep"; date: 
            "2013-12-26- 26"; volume: "5"; issue: "6"; pages: "1704-13"
COMMENT     Sequence Label: LT3REVIN
FEATURES             Location/Qualifiers
     source          1..10319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             23..67
                     /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    334..451
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        469..847
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    477..495
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    513..531
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    549..567
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    585..603
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    621..639
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    657..675
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    693..711
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     756..780
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             871..1548
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(1051..1071)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1463..1486
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           1732..1746
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        1925..2424
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1960..1983)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2357..2376
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(2447..2449,2450..2452,2453..3166)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2492..2513)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2753..2772)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3100..3121
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3183..3756
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3350..3367)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3577..3596
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3772..4566
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3772..4566
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3826..3845)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4436..4455
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    4596..5184
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4649..4669)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5067..5078)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5391..5624
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5696..5817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5733..5752)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5787..5806
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5836..5971
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5991..6010)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5992..6010)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6020..6037)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6020..6036)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6029..6051)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6178..6633
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6265..6284)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6475..6496
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6659..6763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6764..6832,6833..7624)
                     /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     complement(6982..7001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7795..8383
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8284..8303
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8537..8554
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8671..8692
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8707..8712,8713..8730,8731..8737)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8745..8761
                     /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)."
     primer_bind     8750..8772
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8769..8785
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8769..8785
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8804..8824
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8806..8824
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        8852..9078
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             9079..9259
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    9306..9431
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    9924..10157
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
       61 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
      121 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
      181 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
      241 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
      301 attagtgaac ggatctcgac ggtatcggtt aacttttaaa agaaaagggg ggattggggg
      361 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
      421 acaaaaacaa attacaaaaa ttcaaaattt tatcgatcct ctagattcga gtttactccc
      481 tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag aacgtatgca
      541 gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta tcagtgatag
      601 agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca gtttactccc
      661 tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag aacgtataag
      721 ctttaggcgt gtacggtggg cgcctataaa agcagagctc gtttagtgaa ccgtcagatc
      781 gcctggagca attccacaac acttttgtct tataccaact ttccgtacca cttcctaccc
      841 tcgtaaagtc gagcttgcgt tggatccacc atggcctcct ccgagaacgt catcaccgag
      901 ttcatgcgct tcaaggtgcg catggagggc accgtgaacg gccacgagtt cgagatcgag
      961 ggcgagggcg agggccgccc ctacgagggc cacaacaccg tgaagctgaa ggtgaccaag
     1021 ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttccagta cggctccaag
     1081 gtgtacgtga agcaccccgc cgacatcccc gactacaaga agctgtcctt ccccgagggc
     1141 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggcgaccgt gacccaggac
     1201 tcctccctgc aggacggctg cttcatctac aaggtgaagt tcatcggcgt gaacttcccc
     1261 tccgacggcc ccgtgatgca gaagaagacc atgggctggg aggcctccac cgagcgcctg
     1321 tacccccgcg acggcgtgct gaagggcgag acccacaagg ccctgaagct gaaggacggc
     1381 ggccactacc tggtggagtt caagtccatc tacatggcca agaagcccgt gcagctgccc
     1441 ggctactact acgtggacgc caagctggac atcacctccc acaacgagga ctacaccatc
     1501 gtggagcagt acgagcgcac cgagggccgc caccacctgt tcctgtagcg gccgcctcga
     1561 ctagggataa cagggtaatt gtttgaatga ggcttcagta ctttacagaa tcgttgcctg
     1621 cacatcttgg aaacacttgc tgggattact tcgacttctt aacccaacag aaggctcgag
     1681 aaggtatatt gctgttgaca gtgagcgcag gaattataat gcttatctat agtgaagcca
     1741 cagatgtata gataagcatt ataattccta tgcctactgc ctcggacttc aaggggctag
     1801 aattcgagca attatcttgt ttactaaaac tgaatacctt gctatctctt tgatacattt
     1861 ttacaaagct gaattaaaat ggtataaatt aaatcacttt tttcaattga cgcgtaattc
     1921 taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg
     1981 ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt
     2041 aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta
     2101 gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta
     2161 gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc
     2221 ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
     2281 aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg
     2341 tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc
     2401 tcttcctcat ctccgggcct ttcgacctgc agcccaagct tccaccatgg tgagcaaggg
     2461 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     2521 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     2581 gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     2641 gggctacggc ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
     2701 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     2761 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     2821 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2881 ctacaacagc cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa
     2941 cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca
     3001 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca
     3061 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3121 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaagaat tgttcaattc
     3181 cgcccctctc cccccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     3241 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc
     3301 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     3361 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     3421 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     3481 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     3541 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     3601 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     3661 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
     3721 ggggacgtgg ttttcctttg aaaaacacga tgataagctt accggtccac catgattgaa
     3781 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3841 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3901 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     3961 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4021 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4081 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4141 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4201 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4261 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     4321 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4381 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4441 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4501 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4561 ttctgatgta cgatgcatcg aaacgcgtct ggaacaatca acctctggat tacaaaattt
     4621 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     4681 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     4741 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     4801 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     4861 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     4921 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     4981 tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc
     5041 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     5101 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     5161 tctccctttg ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc tagaaaaaca
     5221 tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc
     5281 acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac
     5341 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     5401 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     5461 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     5521 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     5581 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca
     5641 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     5701 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5761 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5821 tgtctggctc tagctatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     5881 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     5941 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     6001 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     6061 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     6121 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     6181 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     6241 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     6301 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     6361 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     6421 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     6481 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     6541 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     6601 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     6661 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     6721 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     6781 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6841 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6901 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6961 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     7021 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     7081 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     7141 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     7201 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     7261 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     7321 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     7381 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     7441 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     7501 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     7561 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     7621 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     7681 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7741 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7801 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7861 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7921 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7981 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     8041 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     8101 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     8161 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     8221 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     8281 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     8341 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     8401 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     8461 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     8521 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     8581 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     8641 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     8701 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     8761 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
     8821 aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct
     8881 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
     8941 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
     9001 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
     9061 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     9121 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     9181 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     9241 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     9301 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     9361 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     9421 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     9481 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     9541 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     9601 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     9661 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     9721 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc
     9781 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     9841 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     9901 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     9961 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
    10021 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
    10081 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
    10141 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
    10201 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
    10261 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattga
//
