LOCUS       sequence-437717-       10273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To inducibly knockdown Rb1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10273)
  AUTHORS   Magrath JW, Sampath SS, Flinchum DA, Hartono AB, Goldberg IN, 
            Boehling JR, Savkovic SD, Lee SB
  TITLE     Comprehensive Transcriptomic Analysis of EWSR1::WT1 Targets 
            Identifies CDK4/6 Inhibitors as an Effective Therapy for 
            Desmoplastic Small Round Cell Tumors.
  JOURNAL   Cancer Res. 2024 May 2;84(9):1426-1442. doi: 
            10.1158/0008-5472.CAN-23-3334.
  PUBMED    38588409
REFERENCE   2  (bases 1 to 10273)
  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: "10.1158/0008-5472";
            journalName: "Cancer Res"; date: "2024-05-2- 2"; volume: "84"; 
            issue: "9"; pages: "1426-1442"
COMMENT     Sequence Label: LT3GEPIR-Rb1shRNA1
FEATURES             Location/Qualifiers
     source          1..10273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..31
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    148..169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(184..189,190..207,208..214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    222..238
                     /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     227..249
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     246..262
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     246..262
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     281..301
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        283..301
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        329..555
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             556..736
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    783..908
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1401..1634
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1819..1863
                     /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    2130..2247
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2263..2641
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    2271..2289
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2307..2325
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2343..2361
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2379..2397
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2415..2433
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2451..2469
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2487..2505
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2550..2574
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(2665..2667,2668..2670,2671..3384)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2710..2731)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2971..2990)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3318..3339
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           3578..3592
                     /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        3772..4271
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3807..3830)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4204..4223
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4292..4891
                     /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(4292..4311)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4788..4808
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4952..5519
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4969..5521
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5113..5130)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5340..5359
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(5533..6150,6151..6156,6157..6192,6193..6195,6196..6231,
                     6232..6234,6235..6270,6271..6279)
                     /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"
     misc_feature    6325..6913
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6378..6398)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6796..6807)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7120..7353
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7425..7546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7462..7481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7516..7535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7586..7721
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7741..7760)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7742..7760)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7770..7787)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7770..7786)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7779..7801)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7928..8383
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8015..8034)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8225..8246
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8409..8513
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8514..8582,8583..9374)
                     /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(8732..8751)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9545..10133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10034..10053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
       61 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      121 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      181 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      241 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
      301 ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc
      361 aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc
      421 cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg
      481 acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta
      541 gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc
      601 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
      661 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
      721 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
      781 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
      841 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
      901 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
      961 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
     1021 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     1081 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     1141 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     1201 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc
     1261 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     1321 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     1381 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     1441 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     1501 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     1561 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     1621 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     1681 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     1741 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     1801 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     1861 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     1921 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     1981 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
     2041 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
     2101 gtgaacggat ctcgacggta tcggttaact tttaaaagaa aaggggggat tggggggtac
     2161 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     2221 aaacaaatta caaaaattca aaattttatc gattctagat tcgagtttac tccctatcag
     2281 tgatagagaa cgtatgaaga gtttactccc tatcagtgat agagaacgta tgcagacttt
     2341 actccctatc agtgatagag aacgtataag gagtttactc cctatcagtg atagagaacg
     2401 tatgaccagt ttactcccta tcagtgatag agaacgtatc tacagtttac tccctatcag
     2461 tgatagagaa cgtatatcca gtttactccc tatcagtgat agagaacgta taagctttag
     2521 gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca gatcgcctgg
     2581 agcaattcca caacactttt gtcttatacc aactttccgt accacttcct accctcgtaa
     2641 agtcgagctt gcgttggatc caccatggtg agcaagggcg aggagctgtt caccggggtg
     2701 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtctggc
     2761 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2821 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2881 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2941 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3001 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3061 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3121 atcatggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc
     3181 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3241 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3301 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3361 ggcatggacg agctgtacaa gtaatgacgt acggcggccg cctcgactag ggataacagg
     3421 gtaattgttt gaatgaggct tcagtacttt acagaatcgt tgcctgcaca tcttggaaac
     3481 acttgctggg attacttcga cttcttaacc caacagaagg ctcgagaagg tatattgctg
     3541 ttgacagtga gcgacctccc atgttgctca aagaatagtg aagccacaga tgtattcttt
     3601 gagcaacatg ggagggtgcc tactgcctcg gacttcaagg ggtctagaat tcgagcaatt
     3661 atcttgttta ctaaaactga ataccttgct atctctttga tacattttta caaagctgaa
     3721 ttaaaatggt ataaattaaa tcactttttt caattgacgc gtaattctac cgggtagggg
     3781 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     3841 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     3901 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     3961 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     4021 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     4081 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     4141 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     4201 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     4261 cgggcctttc gacctgcagc ccaagcttac catgaccgag tacaagccca cggtgcgcct
     4321 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     4381 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     4441 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     4501 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     4561 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     4621 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     4681 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     4741 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     4801 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     4861 ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac
     4921 cgaaaggagc gcacgacccc atgcatccaa ttccgccccc cccccctaac gttactggcc
     4981 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     5041 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     5101 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     5161 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     5221 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     5281 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     5341 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     5401 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     5461 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     5521 atggccacaa ccatgagtag actggacaag agcaaagtca taaacggagc tctggaatta
     5581 ctcaatggtg tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt
     5641 gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctgcca
     5701 atcgagatgc tggacaggca tcatacccac ttctgccccc tggaaggcga gtcatggcaa
     5761 gactttctgc ggaacaacgc caagtcatac cgctgtgctc tcctctcaca tcgcgacggg
     5821 gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag
     5881 ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc
     5941 gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt agcaaaagag
     6001 gaaagagaga cacctaccac cgattctatg cccccacttc tgagacaagc aattgagctg
     6061 ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc
     6121 ctggagaaac agctaaagtg cgaaagcggc gggccgaccg acgcccttga cgattttgac
     6181 ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac
     6241 gctcttgacg attttgacct tgacatgctc cccgggtaat ccggactcga cctgcagcca
     6301 agcttatcga aacgcgtctg gaacaatcaa cctctggatt acaaaatttg tgaaagattg
     6361 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     6421 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     6481 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     6541 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     6601 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     6661 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag
     6721 ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     6781 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     6841 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     6901 gccgcctccc cgcctggaat taattctgca gtcgagacct agaaaaacat ggagcaatca
     6961 caagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg
     7021 aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag
     7081 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     7141 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     7201 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     7261 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     7321 gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc
     7381 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag
     7441 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     7501 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct
     7561 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     7621 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     7681 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
     7741 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     7801 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     7861 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     7921 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     7981 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     8041 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     8101 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     8161 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     8221 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     8281 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     8341 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     8401 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     8461 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     8521 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     8581 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     8641 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     8701 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     8761 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     8821 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     8881 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     8941 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     9001 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     9061 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     9121 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     9181 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     9241 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     9301 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     9361 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     9421 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     9481 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     9541 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     9601 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     9661 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     9721 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     9781 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     9841 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     9901 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     9961 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
    10021 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
    10081 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
    10141 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
    10201 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
    10261 ctcgccgcag ccg
//
