LOCUS       sequence-342817-       10951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for mammalian expression of TomKat 
            (tdTomato-tdKatushka) FRET sensor for Cdc42 activity anchored to the
            membrane with the polybasic and CAAX motif from Kras..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10951)
  AUTHORS   Bell GRR, Rincon E, Akdogan E, Collins SR
  TITLE     Optogenetic control of receptors reveals distinct roles for actin- 
            and Cdc42-dependent negative signals in chemotactic signal 
            processing.
  JOURNAL   Nat Commun. 2021 Nov 16;12(1):6148. doi: 10.1038/s41467-021-26371-z.
  PUBMED    34785668
REFERENCE   2  (bases 1 to 10951)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-11-16"; volume: "12(1):6148. doi"; pages: " 
            10.1038/s41467-021-26371-z"
COMMENT     Sequence Label: pPB_Cdc42-TomKat-sensor-CAAX
FEATURES             Location/Qualifiers
     source          1..10951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1011..1390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1392..1667
                     /label=chicken beta-actin promoter
     intron          1668..2684
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2608..2630
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2692..2711
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2765..2774
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2771..3475
                     /codon_start=1
                     /product="improved version of the oligomeric far-red 
                     fluorescent protein TurboFP635/Katushka (Luker et al., 
                     2015)"
                     /label=Katushka2S
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHAQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     CDS             2771..3475
                     /codon_start=1
                     /product="far-red-shifted derivative of red fluorescent 
                     protein from Entacmaea quadricolor, also known as Katushka 
                     (Shcherbo et al., 2007)"
                     /label=TurboFP635
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHAQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     CDS             3545..4249
                     /codon_start=1
                     /product="improved version of the oligomeric far-red 
                     fluorescent protein TurboFP635/Katushka (Luker et al., 
                     2015)"
                     /label=Katushka2S
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHAQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     CDS             3545..4249
                     /codon_start=1
                     /product="far-red-shifted derivative of red fluorescent 
                     protein from Entacmaea quadricolor, also known as Katushka 
                     (Shcherbo et al., 2007)"
                     /label=TurboFP635
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHAQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     CDS             4517..4864
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             5405..6832
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     misc_feature    7015..7564
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7159..7176)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7386..7405
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(8035..8052)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8041..8265
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(8312..8328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8336..8352
                     /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(8360..8366,8367..8384,8385..8390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8405..8426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   8660..8722
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8763..8783)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8763..8781)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8802..8818)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8826..8842
                     /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(8850..8856,8857..8874,8875..8880))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8895..8916
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9033..9050)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9204..9792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9284..9303)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9963..10754,10755..10823))
                     /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     10586..10605
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10824..10928)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1201 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1381 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     1441 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggsggg
     1501 gggggggggg gsgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
     1561 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
     1621 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     1681 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1741 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1801 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
     1861 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
     1921 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
     1981 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
     2041 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
     2101 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
     2161 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
     2221 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     2281 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     2341 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     2401 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     2461 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     2521 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     2581 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2641 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2701 tggttattgt gctgtctcat cattttggca aagaattggg atccgtccgc tagcgctacc
     2761 ggtcgccacc atggtgggtg aggatagcgt gctgatcacc gagaacatgc acatgaaact
     2821 gtacatggag ggcaccgtga acgaccacca cttcaagtgc acatccgagg gcgaaggcaa
     2881 gccctacgag ggcacccaga ccatgaagat caaggtggtc gagggcggcc ctctcccctt
     2941 cgccttcgac atcctggcta ccagcttcat gtacggcagc aaaaccttta tcaaccacac
     3001 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagaggat
     3061 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccagaacgg
     3121 ctgcctcatc tacaacgtca agatcaacgg ggtgaacttc ccatccaacg gccctgtgat
     3181 gcagaagaaa acactcggct gggaggccag caccgagatg ctgtaccccg ctgacagcgg
     3241 cctgagaggc catgcccaga tggccctgaa gctcgtgggc gggggctacc tgcactgctc
     3301 cctcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcttcta
     3361 cttcgtggac aggagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca
     3421 gcacgagatg gctgtggcca ggtactgcga cctgcctagc aaactggggc acagcgggca
     3481 tggcaccggc agcaccggca gcggcagctc cggtaccgcc tcctccgagg acaacaacat
     3541 ggccatggtg ggtgaggata gcgtgctgat caccgagaac atgcacatga aactgtacat
     3601 ggagggcacc gtgaacgacc accacttcaa gtgcacatcc gagggcgaag gcaagcccta
     3661 cgagggcacc cagaccatga agatcaaggt ggtcgagggc ggccctctcc ccttcgcctt
     3721 cgacatcctg gctaccagct tcatgtacgg cagcaaaacc tttatcaacc acacccaggg
     3781 catccccgac ttctttaagc agtccttccc tgagggcttc acatgggaga ggatcaccac
     3841 atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccaga acggctgcct
     3901 catctacaac gtcaagatca acggggtgaa cttcccatcc aacggccctg tgatgcagaa
     3961 gaaaacactc ggctgggagg ccagcaccga gatgctgtac cccgctgaca gcggcctgag
     4021 aggccatgcc cagatggccc tgaagctcgt gggcgggggc tacctgcact gctccctcaa
     4081 gaccacatac agatccaaga aacccgctaa gaacctcaag atgcccggct tctacttcgt
     4141 ggacaggagg ctggaaagaa tcaaggaggc cgacaaagag acctacgtcg agcagcacga
     4201 gatggctgtg gccaggtact gcgacctgcc tagcaaactg gggcacagcc tcgagaaaga
     4261 gaaagagcgg ccagagattt ctctcccttc agattttgaa cacacaattc atgtcggttt
     4321 tgatgctgtc acaggggagt ttacgggaat gccagagcag tgggcccgct tgcttcagac
     4381 atcaaatatc actaagtcgg agcagaagaa aaacccgcag gctgttctgg atgtgttgga
     4441 gttttacaac tcgaagaaga catccaacag ccagaaatac atgagcttta cagataagtc
     4501 agcttccgga ggaaccagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctgg
     4561 tggtagtgct ggtggttccg gcagtgctgg tggtagtgct ggtggtagta ccagtgctgg
     4621 tggtagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gttccggcag
     4681 tgctggtggt agtgctggtg gtagtaccag tgctggtggt agtgctggtg gtagtgctgg
     4741 tggtagtgct ggtggtagtg ctggtggttc cggcagtgct ggtggtagtg ctggtggtag
     4801 taccagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctgg
     4861 tggtacccag acaattaagt gtgttgttgt gggcgatggt gctgttggta aaacatgtct
     4921 cctgatatcc tacacaacaa acaaatttcc atcggaatat gtaccgactg tttttgacaa
     4981 ctatgcagtc acagttatga ttggtggaga accatatact cttggacttt ttgatactgc
     5041 agggcaagag gattatgaca gattacgacc gctgagttat ccacaaacag atgtatttct
     5101 agtctgtttt tcagtggtct ctccatcttc atttgaaaac gtgaaagaaa agtgggtgcc
     5161 tgagataact caccactgtc caaagactcc tttcttgctt gttgggactc aaattgatct
     5221 cagagatgac ccctctacta ttgagaaact tgccaagaac aaacagaagc ctatcactcc
     5281 agagactgct gaaaagctgg cccgtgacct gaaggctgtc aagtatgtgg agtgttctgc
     5341 acttacacag agaggtctga agaatgtgtt tgatgaggct atcctagctg ccggcggccg
     5401 ctcaatggtg agcaagggcg aggaggtcat caaagagttc atgcgcttca aggtgcgcat
     5461 ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     5521 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     5581 ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga
     5641 catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     5701 cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct
     5761 gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa
     5821 gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa
     5881 gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa
     5941 gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa
     6001 gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga
     6061 gggccgccac cacctgttcc tggggcatgg caccggcagc accggcagcg gcagctccgg
     6121 caccgcctcc tccgaggaca acaacatggc cgtcatcaaa gagttcatgc gcttcaaggt
     6181 gcgcatggag ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg
     6241 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt
     6301 cgcctgggac atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc
     6361 cgccgacatc cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     6421 gatgaacttc gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg
     6481 cacgctgatc tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat
     6541 gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt
     6601 gctgaagggc gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga
     6661 gttcaagacc atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga
     6721 caccaagctg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg
     6781 ctccgagggc cgccaccacc tgttcctgta cggcatggac gagctgtaca agggagcgac
     6841 tctggatcca gatcatgaat tctgcaagat gagcaaagat ggtaaaaaga agaaaaagaa
     6901 gtcaaagaca aagtgtgtaa ttatgtaagt cgacgggccg cggtaacaat tgttaactaa
     6961 cttaagctag caacggtttc cctctagcgg gatcaattcc gccccccccc cctaacgtta
     7021 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     7081 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     7141 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     7201 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     7261 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     7321 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     7381 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     7441 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
     7501 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     7561 aataccatgg tcatgaaaac atttaacatt tctcaacaag atctagaatt agtagaagta
     7621 gcgacagaga agattacaat gctttatgag gataataaac atcatgtggg agcggcaatt
     7681 cgtacgaaaa caggagaaat catttcggca gtacatattg aagcgtatat aggacgagta
     7741 actgtttgtg cagaagccat tgcgattggt agtgcagttt cgaatggaca aaaggatttt
     7801 gacacgattg tagctgttag acacccttat tctgacgaag tagatagaag tattcgagtg
     7861 gtaagtcctt gtggtatgtg tagggagttg atttcagact atgcaccaga ttgttttgtg
     7921 ttaatagaaa tgaatggcaa gttagtcaaa actacgattg aagaactcat tccactcaaa
     7981 tatacccgaa attaaaagtc gagatgcatc gatgatctag agctcgctga tcagcctcga
     8041 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     8101 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     8161 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     8221 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     8281 gaacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     8341 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     8401 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     8461 gaaacctgtc gtgccagcgg atcttaaaag ttttgttact ttatagaaga aattttgagt
     8521 ttttgttttt ttttaataaa taaataaaca taaataaatt gtttgttgaa tttattatta
     8581 gtatgtaagt gtaaatataa taaaacttaa tatctattca aattaataaa taaacctcga
     8641 tatacagacc gataaaacac atgcgtcaat tttacgcatg attatcttta acgtacgtca
     8701 caatatgatt atctttctag ggttaatcta gtatacgcgt ttaattaatc cagcttttgt
     8761 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     8821 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     8881 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     8941 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     9001 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     9061 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     9121 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     9181 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     9241 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     9301 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     9361 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     9421 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     9481 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     9541 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     9601 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     9661 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     9721 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     9781 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     9841 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     9901 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     9961 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
    10021 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
    10081 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
    10141 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
    10201 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
    10261 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
    10321 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
    10381 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
    10441 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
    10501 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
    10561 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
    10621 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
    10681 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
    10741 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
    10801 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
    10861 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
    10921 gttccgcgca catttccccg aaaagtgcca c
//
