LOCUS       sequence-273129-       10217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses lysosomal-METRIQ (DNAse II-sfGFP together with cytosolic 
            RFP) to measure lysosomal activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10217)
  AUTHORS   Ishii S, Matsuura A, Itakura E
  TITLE     Identification of a factor controlling lysosomal homeostasis using a
            novel lysosomal trafficking probe.
  JOURNAL   Sci Rep. 2019 Aug 12;9(1):11635. doi: 10.1038/s41598-019-48131-2.
  PUBMED    31406169
REFERENCE   2  (bases 1 to 10217)
  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: "Sci Rep."; 
            date: "2019-08-12"; volume: "9(1):11635. doi"; pages: " 
            10.1038/s41598-019-48131-2"
COMMENT     Sequence Label: pCW57.1 Lysosomal-METRIQ
FEATURES             Location/Qualifiers
     source          1..10217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        212..526
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    220..238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    256..274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    291..309
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    327..345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    363..381
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    398..416
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    434..452
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     499..523
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1648..2358
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             1648..2358
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1675..1703)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2298..2318
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2383..2436
                     /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             2437..3141
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2579..2597)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2847..2866
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        3159..3669
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3588..3606
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3679..4275
                     /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(3679..3698)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4175..4195
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4276..4329
                     /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             join(4330..4947,4948..4953,4954..4989,4990..4992,4993..5028,
                     5029..5031,5032..5067,5068..5076)
                     /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"
     primer_bind     complement(5116..5136)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5534..5545)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5750..5983
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6055..6176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6092..6111)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6146..6165
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6216..6351
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        6351..6422
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6423..6491,6492..7283)
                     /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(6641..6660)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7454..8042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7943..7962
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8196..8213
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8330..8351
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8366..8371,8372..8389,8390..8396)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8404..8420
                     /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     8409..8431
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8428..8444
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8428..8444
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8463..8483
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8465..8483
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        8511..8737
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             8738..8918
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    8965..9090
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    9583..9816
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             10001..10045
                     /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"
ORIGIN
        1 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac
      241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
      301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
      421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
      481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggacc
      541 atgggcatcc cgctgctgct ggcagcgctg ctgtgcgtcc ccgccggggc cctgacctgc
      601 tacggggact ccgggcagcc tgtagactgg ttcgtggtct acaagctgcc agctcttaga
      661 gggtccgggg aggcggcgca gagagggctg cagtacaagt atctggacga gagctccgga
      721 ggctggcggg acggcagggc actcatcaac agcccggagg gggccgtggg ccgaagcctg
      781 cagccgctgt accggagcaa caccagccag ctcgccttcc tgctctacaa tgaccaaccg
      841 cctcaaccca gcaaggctca ggactcttcc atgcgtgggc acacgaaggg tgtcctgctc
      901 cttgaccacg atgggggctt ctggctggtc cacagtgtac ctaacttccc tccaccggcc
      961 tcctctgctg catacagttg gcctcatagc gcctgtacct acgggcagac cctgctctgt
     1021 gtgtcttttc ccttcgctca gttctcgaag atgggcaagc agctgaccta cacctacccc
     1081 tgggtctata actaccagct ggaagggatc tttgcccagg aattccccga cttggagaat
     1141 gtggtcaagg gccaccacgt tagccaagaa ccctggaaca gcagcatcac actcacatcc
     1201 caggccgggg ctgttttcca gagctttgcc aagttcagca aatttggaga tgacctgtac
     1261 tccggctggt tggcagcagc ccttggtacc aacctgcagg tccagttctg gcacaaaact
     1321 gtaggcatcc tgccctctaa ctgctcggat atctggcagg ttctgaatgt gaaccagata
     1381 gctttccctg gaccagccgg cccaagcttc aacagcacag aggaccactc caaatggtgc
     1441 gtgtccccaa aagggccctg gacctgcgtg ggtgacatga atcggaacca gggagaggag
     1501 caacggggtg ggggcacact gtgtgcccag ctgccagccc tctggaaagc cttccagccg
     1561 ctggtgaaga actaccagcc ctgtaatggc atggccagga agcccagcag agcttataag
     1621 atcgttaacg gtggaggtgg aagcggtagc aaaggagaag aacttttcac tggagttgtc
     1681 ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt ccgtggagag
     1741 ggtgaaggtg atgctacaaa cggaaaactc acccttaaat ttatttgcac tactggaaaa
     1801 ctacctgttc cgtggccaac acttgtcact actctgacct atggtgttca atgcttttcc
     1861 cgttatccgg atcacatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat
     1921 gtacaggaac gcactatatc tttcaaagat gacgggacct acaagacgcg tgctgaagtc
     1981 aagtttgaag gtgataccct tgttaatcgt atcgagttaa agggtattga ttttaaagaa
     2041 gatggaaaca ttcttggaca caaactcgag tacaacttta actcacacaa tgtatacatc
     2101 acggcagaca aacaaaagaa tggaatcaaa gctaacttca aaattcgcca caacgttgaa
     2161 gatggttccg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct
     2221 gtccttttac cagacaacca ttacctgtcg acacaatctg tcctttcgaa agatcccaac
     2281 gaaaagcgtg accacatggt ccttcttgag tttgtaactg ctgctgggat tacacatggc
     2341 atggatgagc tctacaaagg tggaggtgga tccggcagtg gagagggcag aggaagtctg
     2401 ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggaggataac
     2461 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     2521 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     2581 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     2641 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     2701 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     2761 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     2821 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     2881 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     2941 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     3001 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     3061 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc
     3121 ggcatggacg agctgtacaa ggaattctct tgagatccgg ggttggggtt gcgccttttc
     3181 caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc
     3241 agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc
     3301 ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa
     3361 ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tctcactagt
     3421 accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg
     3481 gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg
     3541 gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc
     3601 tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc
     3661 tctccccagc aattcaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     3721 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     3781 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     3841 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     3901 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     3961 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     4021 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     4081 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     4141 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     4201 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     4261 cgcaagcccg gtgccgaagg tagaggttct ctcctcactt gtggtgatgt tgaagaaaac
     4321 cctggtccaa tgtctagact ggacaagagc aaagtcataa acggagctct ggaattactc
     4381 aatggtgtcg gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct gggagttgag
     4441 cagcctaccc tgtactggca cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc
     4501 gagatgctgg acaggcatca tacccacttc tgccccctgg aaggcgagtc atggcaagac
     4561 tttctgcgga acaacgccaa gtcataccgc tgtgctctcc tctcacatcg cgacggggct
     4621 aaagtgcatc tcggcacccg cccaacagag aaacagtacg aaaccctgga aaatcagctc
     4681 gcgttcctgt gtcagcaagg cttctccctg gagaacgcac tgtacgctct gtccgccgtg
     4741 ggccacttta cactgggctg cgtattggag gaacaggagc atcaagtagc aaaagaggaa
     4801 agagagacac ctaccaccga ttctatgccc ccacttctga gacaagcaat tgagctgttc
     4861 gaccggcagg gagccgaacc tgccttcctt ttcggcctgg aactaatcat atgtggcctg
     4921 gagaaacagc taaagtgcga aagcggcggg ccgaccgacg cccttgacga ttttgactta
     4981 gacatgctcc cagccgatgc ccttgacgac tttgaccttg atatgctgcc tgctgacgct
     5041 cttgacgatt ttgaccttga catgctcccc gggtaactaa gtaaggatcg atccaagata
     5101 tcgtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     5161 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     5221 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     5281 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     5341 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     5401 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct
     5461 gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     5521 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     5581 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     5641 cgcctccccg cctgtttcgc ctcggcgtcc ggactagagg tacctttaag accaatgact
     5701 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     5761 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     5821 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     5881 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     5941 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat
     6001 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg
     6061 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     6121 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     6181 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     6241 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     6301 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctttcaaata
     6361 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     6421 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     6481 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6541 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6601 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6661 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6721 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6781 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6841 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6901 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6961 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     7021 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     7081 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     7141 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     7201 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     7261 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     7321 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7381 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     7441 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     7501 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     7561 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     7621 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7681 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     7741 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     7801 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     7861 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     7921 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7981 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     8041 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     8101 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     8161 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     8221 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     8281 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     8341 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     8401 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     8461 gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta
     8521 tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg
     8581 agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt
     8641 aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag
     8701 agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat
     8761 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     8821 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     8881 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
     8941 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
     9001 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
     9061 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
     9121 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
     9181 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
     9241 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
     9301 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
     9361 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
     9421 cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     9481 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     9541 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     9601 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     9661 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     9721 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     9781 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     9841 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     9901 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     9961 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
    10021 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
    10081 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
    10141 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
    10201 cccaaccccg aggggac
//
