LOCUS       sequence-462153-        9450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid containing TRE-pfv-Sapphire for the 
            doxycycline-inducible expression of genetically encoded multimeric 
            nanoparticles (GEM).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9450)
  AUTHORS   Korunova E, Sikirzhystki V, Twiss JL, Vasquez P, Shtutman M
  TITLE     Single Particle Tracking of Genetically Encoded Nanoparticles: 
            Optimizing Expression for Cytoplasmic Diffusion Studies.
  JOURNAL   bioRxiv [Preprint]. 2024 Nov 18:2024.11.17.623896. doi: 
            10.1101/2024.11.17.623896.
  PUBMED    39605363
REFERENCE   2  (bases 1 to 9450)
  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: "bioRxiv 
            [Preprint]. 2024 Nov 18:2024.11.17.623896. doi: 
            10.1101/2024.11.17.623896."
COMMENT     Sequence Label: TRE-pfv-Sapphire
FEATURES             Location/Qualifiers
     source          1..9450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    103..224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(114..133)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     168..187
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             296..529
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             734..745
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1143..1163
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(1203..1211,1212..1247,1248..1250,1251..1286,
                     1287..1289,1290..1325,1326..1331,1332..1949))
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(1950..2003)
                     /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             complement(2004..2600)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2084..2104)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2581..2600
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(2610..3120)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2673..2691)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             complement(3130..3147)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3155..3871)
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     promoter        complement(4986..5300)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(4989..5013)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5060..5078
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5096..5114
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5131..5149
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5167..5185
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5203..5221
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5238..5256
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5274..5292
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    5354..5471
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(5684..5728)
                     /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    5913..6146
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6639..6764
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6811..6991
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(6992..7218)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(7246..7266)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7246..7264)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7285..7301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7285..7301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7298..7320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7309..7325
                     /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(7333..7339,7340..7357,7358..7363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7378..7399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7516..7533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7687..8275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7767..7786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8446..9237,9238..9306))
                     /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     9069..9088
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9307..9378)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(9378..9450,1..63)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg
       61 gatgggcgga gttaggggcg ggatagctag agccagacat gataagatac attgatgagt
      121 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
      181 ctattgcttt atttgtaacc attataagct gcaataaaca agttcctctc actctctgat
      241 attcatttct ttgcaagtta taaatactga ataataagat gacatgaact actactgcta
      301 gagattttcc acactgacta aaagggtctg agggatctct agttaccaga gtcacacaac
      361 agacgggcac acactacttg aagcactcaa ggcaagcttt attgaggctt aagcagtggg
      421 ttccctagtt agccagagag ctcccaggct cagatctggt ctaaccagag agacccagta
      481 caagcaaaaa gcagatcttg tcttcgttgg gagtgaatta gcccttccag tccccccttt
      541 tcttttaaaa agtggctaag atctacagct gccttgtaag tcattggtct taaaggtacc
      601 tctagtccgg acgccgaggc gaaacaggcg gggaggcggc ccaaagggag atccgactcg
      661 tctgagggcg aaggcgaaga cgcggaagag gccgcagagc cggcagcagg ccgcgggaag
      721 gaaggtccgc tggattgagg gccgaaggga cgtagcagaa ggacgtcccg cgcagaatcc
      781 aggtggcaac acaggcgagc agccatggaa aggacgtcag cttccccgac aacaccacgg
      841 aattgtcagt gcccaacagc cgagcccctg tccagcagcg ggcaaggcag gcggcgatga
      901 gttccgccgt ggcaataggg agggggaaag cgaaagtccc ggaaaggagc tgacaggtgg
      961 tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac agtgcacacc acgccacgtt
     1021 gcctgacaac gggccacaac tcctcataaa gagacagcaa ccaggattta tacaaggagg
     1081 agaaaatgaa agccatacgg gaagcaatag catgatacaa aggcattaaa gcagcgtatc
     1141 cacatagcgt aaaaggagca acatagttaa gaatacgata tcttggatcg atccttactt
     1201 agttacccgg ggagcatgtc aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca
     1261 aggtcaaagt cgtcaagggc atcggctggg agcatgtcta agtcaaaatc gtcaagggcg
     1321 tcggtcggcc cgccgctttc gcactttagc tgtttctcca ggccacatat gattagttcc
     1381 aggccgaaaa ggaaggcagg ttcggctccc tgccggtcga acagctcaat tgcttgtctc
     1441 agaagtgggg gcatagaatc ggtggtaggt gtctctcttt cctcttttgc tacttgatgc
     1501 tcctgttcct ccaatacgca gcccagtgta aagtggccca cggcggacag agcgtacagt
     1561 gcgttctcca gggagaagcc ttgctgacac aggaacgcga gctgattttc cagggtttcg
     1621 tactgtttct ctgttgggcg ggtgccgaga tgcactttag ccccgtcgcg atgtgagagg
     1681 agagcacagc ggtatgactt ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc
     1741 agggggcaga agtgggtatg atgcctgtcc agcatctcga ttggcagggc atcgagcagg
     1801 gcccgcttgt tcttcacgtg ccagtacagg gtaggctgct caactcccag cttttgagcg
     1861 agtttccttg tcgtcaggcc ttcgataccg acaccattga gtaattccag agctccgttt
     1921 atgactttgc tcttgtccag tctagacatt ggaccagggt tttcttcaac atcaccacaa
     1981 gtgaggagag aacctctacc ttcggcaccg ggcttgcggg tcatgcacca ggtgcgcggt
     2041 ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg
     2101 ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact
     2161 ccggggagca cgacggcgct gcccagaccc ttgccctggt ggtcgggcga gacgccgacg
     2221 gtggccagga accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt
     2281 tgctgcgcgg ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac
     2341 accgcccccg cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc
     2401 gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg
     2461 gtgacccgct cgatgtggcg gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg
     2521 aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc
     2581 gtgggcttgt actcggtcat ggtgaattgc tggggagaga ggtcggtgat tcggtcaacg
     2641 agggagccga ctgccgacgt gcgctccgga ggcttgcaga atgcggaaca ccgcgcgggc
     2701 aggaacaggg cccacactac cgccccacac cccgcctccc gcaccgcccc ttcccggccg
     2761 ctgctctcgg cgcgccctgc tgagcagccg ctattggcca cagcccatcg cggtcggcgc
     2821 gctgccattg ctccctggcg ctgtccgtct gcgagggtac tagtgagacg tgcggcttcc
     2881 gtttgtcacg tccggcacgc cgcgaaccgc aaggaacctt cccgacttag gggcggagca
     2941 ggaagcgtcg ccggggggcc cacaagggta gcggcgaaga tccgggtgac gctgcgaacg
     3001 gacgtgaaga atgtgcgaga cccagggtcg gcgccgctgc gtttcccgga accacgccca
     3061 gagcagccgc gtccctgcgc aaacccaggg ctgccttgga aaaggcgcaa ccccaacccc
     3121 ggatccttag tggtggtggt ggtggtggac cggttcattt gtacaattca tcaataccat
     3181 gggtaatacc agcagcagta acaaattcta acaagaccat gtggtctctc ttttcgtttg
     3241 gatctttgga taaggcagat tgaatggata agtaatggtt gtctggtaac aagactggac
     3301 catcaccaat tggagtattt tgttgataat ggtcagctaa ttgaacagaa ccatcttcaa
     3361 tgttgtgtct aattttgaag ttaactttga taccattctt ttgtttgtca gccatgatgt
     3421 aaacattgtg agagttaaag ttgtattcca atttgtgacc taaaatgtta ccatcttctt
     3481 taaaatcaat accttttaat tcgattctat taactaaggt atcaccttca aacttgactt
     3541 cagctctggt cttgtagtta ccgtcatctt tgaaaaaaat agttctttct tgaacataac
     3601 cttctggcat ggcagacttg aaaaagtcat gttgtttcat atgatctggg tatctagcaa
     3661 aacattgaac accataagaa aaagtagtga ctaaggttgg ccatggaact ggcaatttac
     3721 cagtagtaca aataaatttt aaggtcaatt taccgtaagt agcatcacct tcaccttcac
     3781 cggagacaga aaatttgtga ccattaacat caccatctaa ttcaaccaaa attgggacaa
     3841 caccagtgaa taattcttca cctttagaca tcttaattaa cccggggatc cgtcgaccgg
     3901 agcctgatcc cgacccacta ccgctgccag accactctaa gactactatt gcctctgggt
     3961 tctttattct tattcctata gcttcactaa ccaagaatgc aacgtcgtct ccttcaggtc
     4021 ctagctcttg tagagtcaca tcttggccaa ctacaacgtc aagaacagct ggtgtattgg
     4081 caaatattat taccttattc tcatcaatgt ttagagtaac aatgattcct cccctgaata
     4141 tctcttttag cacttcaact agcatctttc cactcttctc gtaaattttt agcaactttg
     4201 catatctttt tgggttaatt atcagcccaa atggtccagc tgacgcattt gaggcttcct
     4261 gcagtgcgtt cattaaatca ttaagtgctt cttctggatt gtcccaggag gaaagtttca
     4321 ttttcttaat tccctctgtg gatagaattt ctcttattac tagcgactct tcgtctttaa
     4381 ctaggagttc cccagcttta gttgctatag atacatcgta agtcccatcg ttgagttctc
     4441 ttattcccac atagaatttc ttcttcaata atggaattgg cttgtactct tgctttacta
     4501 ccctaacacc atcttcaaat ttaatgatgt caaccctgaa tgattgtcct tctattctcg
     4561 ttactggcaa gtgctttagt aaagatcttc ccttatttac cccatccaaa agagcacttc
     4621 tcaacttctc aaaatattcc acgttagatt cctccttctt gttatcattt atgtgagcct
     4681 caattccagt tagctccttg acttcctcaa atccaccctt cagctcagtt acttgttctg
     4741 gatccaaatt tagaaggagt gccatgaatt ctccaacgtg agccttctcc tcccttgcaa
     4801 catcaaggag aatctttctt acgttttcat cttcggaata tcttgccatc tgctcatata
     4861 gatttattgc atcaagctca gctattattg ctaatctcag aatttccatt aattcttctt
     4921 tggtgtatgg cttatcccta tttataaggg ttggatttat tgagagcatg ctagccaatt
     4981 ctccaggcga tctgacggtt cactaaacga gctctgctta tataggcctc ccaccgtaca
     5041 cgcctacctc gacatacgtt ctctatcact gatagggagt aaactcgaca tacgttctct
     5101 atcactgata gggataaact cgacatacgt tctctatcac tgatagggag taaactcgac
     5161 atacgttctc tatcactgat agggagtaaa ctcgacatac gttctctatc actgataggg
     5221 agtaaactcg acatcgttct ctatcactga tagggagtaa actcgacata cgttctctat
     5281 cactgatagg gagtaaactc gacatatcga ttcgcggcca aagtggatct ctgctgtccc
     5341 tgtaataaac ccgaaaattt tgaatttttg taatttgttt ttgtaattct ttagtttgta
     5401 tgtctgttgc tattatgtct actattcttt cccctgcact gtacccccca atcccccctt
     5461 ttcttttaaa attgtggatg aatactgcca tttgtctcga ggtcgagaat tgtcccctcg
     5521 gggttgggag gtgggtctga aacgataatg gtgaatatcc ctgcctaact ctattcacta
     5581 tagaaagtac agcaaaaact attcttaaac ctaccaagcc tcctactatc attatgaata
     5641 attttatata ccacagccaa tttgttatgt taaaccaatt ccacaaactt gcccatttat
     5701 ctaattccaa taattcttgt tcattctttt cttgctggtt ttgcgattct tcaattaagg
     5761 agtgtattaa gcttgtgtaa ttgttaattt ctctgtccca ctccatccag gtcgtgtgat
     5821 tccaaatctg ttccagagat ttattactcc aactagcatt ccaaggcaca gcagtggtgc
     5881 aaatgagttt tccagagcaa ccccaaatcc ccaggagctg ttgatccttt aggtatcttt
     5941 ccacagccag gattcttgcc tggagctgct tgatgcccca gactgtgagt tgcaacagat
     6001 gctgttgcgc ctcaatagcc ctcagcaaat tgttctgctg ctgcactata ccagacaata
     6061 attgtctggc ctgtaccgtc agcgtcattg acgctgcgcc catagtgctt cctgctgctc
     6121 ccaagaaccc aaggaacaaa gctcctattc ccactgctct tttttctctc tgcaccactc
     6181 ttctctttgc cttggtgggt gctactccta atggttcaat ttttactact ttatatttat
     6241 ataattcact tctccaattg tccctcatat ctcctcctcc aggtctgaag atcagcggcc
     6301 gcttgctgtg cggtggtctt acttttgttt tgctcttcct ctatcttgtc taaagcttcc
     6361 ttggtgtctt ttatctctat cctttgatgc acacaataga gggttgctac tgtattatat
     6421 aatgatctaa gttcttctga tcctgtctga agggatggtt gtagctgtcc cagtatttgt
     6481 ctacagcctt ctgatgtttc taacaggcca ggattaactg cgaatcgttc tagctccctg
     6541 cttgcccata ctatatgttt taatttatat tttttctttc cccctggcct taaccgaatt
     6601 ttttcccatc gcgatctaat tctcccccgc ttaatactga cgctctcgca cccatctctc
     6661 tccttctagc ctccgctagt caaaattttt ggcgtactca ccagtcgccg cccctcgcct
     6721 cttgccgtgc gcgcttcagc aagccgagtc ctgcgtcgag agagctcctc tggtttccct
     6781 ttcgctttca agtccctgtt cgggcgccac tgctagagat tttccacact gactaaaagg
     6841 gtctgaggga tctctagtta ccagagtcac acaacagacg ggcacacact acttgaagca
     6901 ctcaaggcaa gctttattga ggcttaagca gtgggttccc tagttagcca gagagctccc
     6961 aggctcagat ctggtctaac cagagagacc cgtttatgta tcgagctagg cacttaaata
     7021 caatatctct gcaatgcggc aattcagtgg ttcgtccaat ccatgtcaga cccgtctgtt
     7081 gccttcctaa taaggcacga tcgtaccacc ttacttccac caatcggcat gcacggtgct
     7141 ttttctctcc ttgtaaggca tgttgctaac tcatcgttac catgttgcaa gactacaaga
     7201 gtattgcata agactacatt aagcttgcag ctccagcttt tgttcccttt agtgagggtt
     7261 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     7321 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     7381 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     7441 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     7501 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     7561 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     7621 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     7681 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     7741 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     7801 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     7861 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     7921 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     7981 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     8041 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     8101 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     8161 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     8221 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     8281 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     8341 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     8401 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     8461 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     8521 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     8581 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     8641 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     8701 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     8761 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     8821 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     8881 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     8941 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     9001 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     9061 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     9121 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     9181 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     9241 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     9301 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     9361 cggatacata tttgaaaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga
     9421 ggccgaggcg gcctcggcct ctgcataaat
//
