LOCUS       sequence-472028-       14469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EcLeu aaRS/tRNA system for TAG suppression with EGFP containing two 
            stop codons for dual suppression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14469)
  AUTHORS   Osgood AO, Zheng Y, Roy SJS, Biris N, Hussain M, Loynd C, Jewel D, 
            Italia JS, Chatterjee A
  TITLE     An Efficient Opal-Suppressor Tryptophanyl Pair Creates New Routes 
            for Simultaneously Incorporating up to Three Distinct Noncanonical 
            Amino Acids into Proteins in Mammalian Cells.
  JOURNAL   Angew Chem Int Ed Engl. 2023 May 2;62(19):e202219269. doi: 
            10.1002/anie.202219269. Epub 2023 Apr 12.
  PUBMED    36905325
REFERENCE   2  (bases 1 to 14469)
  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: "Angew Chem 
            Int Ed Engl."; date: "2023-05-2"; volume: "62(19):e202219269. doi"; 
            pages: " 10.1002/anie.202219269. Epub 2023 Apr 12"
COMMENT     Sequence Label: pAcBac3-EcLeu TAG-EGFP**
FEATURES             Location/Qualifiers
     source          1..14469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  894..1118
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(1185..1718)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1907..1912,1913..1929,1930..1935))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    2365..2589
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2578..2595
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2633..5224)
                     /codon_start=1
                     /product="aminoacyl-tRNA synthetase specific for the 
                     fluorescent amino acid Anap (Chatterjee et al., 2013)"
                     /label=AnapRS
                     /note="evolved from an E. coli leucyl-tRNA synthetase"
                     /translation="MEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSILPYPS
                     GRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPAEGAAVKNNTAPAPWTYDN
                     IAYMKNQLKMLGFGYDWSRELATCTPEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQ
                     TVLANEQVIDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTVKTMQRN
                     WIGRSEGVEITFNVNDYDNTLTVYTTRPDAFMGCTYLAVAAGHPLAQKAAENNPELAAF
                     IDECRNTKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPG
                     HDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAI
                     ADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDV
                     VMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESSWIYARYTCPQYKEGMLDSEA
                     ANYWLPVDIAIGGIEHAIMGLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYY
                     VGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVE
                     RYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAALNVDAL
                     TENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQDRALMQEA
                     LLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRA
                     KITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVGGPV"
     regulatory      5221..5230
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5268..5287)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5269..5287)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5340..5739)
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     tRNA            complement(join(5825..5871,5872..5874,5875..5908))
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        complement(5913..6007)
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    5913..5931
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5941..5959
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            complement(join(6021..6067,6068..6070,6071..6104))
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        complement(6109..6203)
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    6109..6127
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6137..6155
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            complement(join(6217..6263,6264..6266,6267..6300))
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        complement(6305..6399)
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    6305..6323
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6333..6351
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            complement(join(6413..6459,6460..6462,6463..6496))
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        complement(6501..6595)
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    6501..6519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6529..6547
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            complement(join(6609..6655,6656..6658,6659..6692))
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        complement(6697..6791)
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    6697..6715
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6725..6743
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        6883..7186
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7188..7464
                     /label=chicken beta-actin promoter
     intron          7465..8481
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     8405..8427
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     8489..8508
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      8545..8554
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8551..8553,8554..8556,8557..9267)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT*GKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNV*IMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8596..8617)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(8857..8876)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9204..9225
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             9277..9306
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             9322..9351
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             9325..9354
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             9328..9357
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     misc_feature    9396..9984
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9449..9469)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9867..9878)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10052..10071)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    10117..10172
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(10171..10190)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        10524..10615
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     10540..10557
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             join(10674..10721,10722..12209)
                     /codon_start=1
                     /product="vesicular stomatitis virus G glycoprotein"
                     /label=VSV-G
                     /note="Indiana strain"
                     /translation="MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSD
                     LNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPS
                     VEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQ
                     FINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFR
                     SNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQT
                     SVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLK
                     YFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFP
                     LYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWF
                     SSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK"
     polyA_signal    12342..12476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12379..12398)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12433..12452
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  12505..12670
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      12854..13308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12941..12960)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13150..13171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        13334..13438
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13439..13507,13508..14299)
                     /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(13657..13676)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagacc agccgcgtaa
      841 cctggcaaaa tcggttacgg ttgagtaata aatggatgcc ctgcgtaagc gggtgtgggc
      901 ggacaataaa gtcttaaact gaacaaaata gatctaaact atgacaataa agtcttaaac
      961 tagacagaat agttgtaaac tgaaatcagt ccagttatgc tgtgaaaaag catactggac
     1021 ttttgttatg gctaaagcaa actcttcatt ttctgaagtg caaattgccc gtcgtattaa
     1081 agaggggcgt ggccaagggc atggtaaaga ctatattcgc ggcgttgtga caatttaccg
     1141 aacaactccg cggccgggaa gccgatctcg gcttgaacga attgttaggt ggcggtactt
     1201 gggtcgatat caaagtgcat cacttcttcc cgtatgccca actttgtata gagagccact
     1261 gcgggatcgt caccgtaatc tgcttgcacg tagatcacat aagcaccaag cgcgttggcc
     1321 tcatgcttga ggagattgat gagcgcggtg gcaatgccct gcctccggtg ctcgccggag
     1381 actgcgagat catagatata gatctcacta cgcggctgct caaacttggg cagaacgtaa
     1441 gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat gaatgtctta
     1501 ctacggagca agttcccgag gtaatcggag tccggctgat gttgggagta ggtggctacg
     1561 tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac ttggtcaggg
     1621 ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt tttagggcga
     1681 ctgccctgct gcgtaacatc gttgctgctg cgtaacatcg ttgctgctcc ataacatcaa
     1741 acatcgaccc acggcgtaac gcgcttgctg cttggatgcc cgaggcatag actgtacaaa
     1801 aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt
     1861 caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagtt tacgaaccga
     1921 acaggcttat gtcaactggg ttcgtgcctt catccgtttc cacggtgtgc gtcacccggc
     1981 aaccttgggc agcagcgaag tcgaggcatt tctgtcctgg ctggcgaacg agcgcaaggt
     2041 ttcggtctcc acgcatcgtc aggcattggc ggccttgctg ttcttctacg gcaaggtgct
     2101 gtgcacggat ctgcccttgc ttcaggagat cggtagacct cggccgtcgc ggcgcttgcc
     2161 ggtggtgctg accccggatg aagtggttcg catcctcggt tttctggaag gcgagcatcg
     2221 tttgttcgcc caggactcta gctatagttc tagtggttgg ctacgtaccc gtagtggcta
     2281 tggcagggct tgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     2341 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     2401 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
     2461 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
     2521 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
     2581 aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagactcg agttaaacgg
     2641 gcccgccaac gaccagattg aggagtttac ctggtacgta aatcacttta cgtacagtaa
     2701 cgccatcaag atattttgct accagatgtt cctggccagc acgttcgcga acctgttctt
     2761 ccgttgcgtc caccggaacg gtgattttgg cacggacttt accgttaacc tgcaccacga
     2821 ccagcgtgga gtcttccacc atcgcttttt cgtcagcaac cggccacggc gcgttgtcga
     2881 tatcgccttc gcctttcagt tcctgccaca gcgtgaagca gatgtgcggg gtgaacgggt
     2941 taagcatacg gacaacggcc agcagtgctt cctgcatcag agcgcgatcc tgctcgccat
     3001 cggttggtgc tttcgccagt ttgttcatca gctccataat cgccgcaatt gcggtgttga
     3061 aggtctgacg acggccgata tcatcggtca ctttagcgat cgttttatgc acatcgcgac
     3121 gcagcgcttt ctgattttca gtcagcgcat caacgttcag tgccgcaaca tcaccttttg
     3181 ctgtgtgctc gtaaaccagt ttccagacac gtttcaggaa gcggttagcc ccttccacac
     3241 cggattcctg ccattcgaga gtcatatcag ccggagaagc aaacatcata aacagacgaa
     3301 cggtgtccgc gccgtaacgt tcaaccatca cctgcgggtc gataccgttg ttcttcgact
     3361 tggacatttt gctcatgccg gtataaacca gttcatggcc tgccgcatct ttcgctttca
     3421 cgatacggcc tttctcgtca cgttcaacga tagcatcaac cggggaaacc cagttacgtt
     3481 cgccgttttc gccaacatag tagaaggcat ctgccagcac cataccctga cacagcaact
     3541 gtttcgctgg ttcgtcagag ttcaccatgc ctgcatcacg catcagtttg tggaagaagc
     3601 ggaagtagag cagacccata atggcgtgtt caataccacc aatagcgata tccaccggca
     3661 gccagtagtt agccgcttcg gaatccagca taccttcttt gtactgcggg caagtgtagc
     3721 gcgcatagat ccaggaggac tccataaagg tgtcgaaagt gtcggtttca cgcagtgctg
     3781 gcataccgtt aacggtagtt ttcgcccact ccggatctgc tttaatcggg ctggtaatgc
     3841 cgtccattac cacatcttcc ggcaggatca ccggcagctg gtcgtccggg gtcggcatta
     3901 cggtaccgtc ttctagagtc accatcggaa tcggcgcgcc ccagtaacgc tgacgggaaa
     3961 caccccagtc gcgcaggcgg tagttcactt tacgctcgcc aacgcccatc gcagtcagtt
     4021 tatcggcgat ggcgttgaag gccgcttcat ggtcaagacc gttgaactcg ccagagttga
     4081 acagcacgcc tttttcagtc agggcttgct gagaaagatc tggctcagag ccgtcagctg
     4141 ccaggataac cggtttgatg ttcaggccgt atttagaggc aaactcgtag tcgcgctggt
     4201 cgtgccccgg taccgccata actgcgcccg tgccgtactc catcaatacg aagtttgctg
     4261 cccaaacggg aatttcttcg cccgttaatg ggtgaaccgc tttaaagcca gtatcgacgc
     4321 cttttttctc catcgtcgcc atttcagctt cggcaacttt ggtgttacgg cattcgtcaa
     4381 taaaggccgc cagttcagga ttattttccg ccgctttctg cgccagcgga tgacccgcag
     4441 ctaccgccag gtaggtacaa cccataaacg cgtccgggcg ggtagtgtaa acggtcagcg
     4501 tgttgtcata gtcgttaacg ttgaaggtga tctccacgcc ttcggaacga ccgatccagt
     4561 tacgctgcat ggttttaacg gtgtctggcc agtgatccag tttatccaga tcgttgagca
     4621 gctcgtcagc gtaagcagtg attttgataa accactgcgg gatctcttta cgttcaactt
     4681 tggtatcgca gcgccagcag cagccgtcga taacttgttc gttcgccagt acggtctggt
     4741 cgttcggaca ccagttgacc gcagaagtct tcttatatac caggcctttt ttatacagct
     4801 cggtgaagaa tttctgttcc caacggtagt attccggcgt acaggttgcc agctcgcggc
     4861 tccagtcata accaaagccc agcattttga gctggttttt catatacgcg atgttgtcgt
     4921 acgtccacgg tgccggagcg gtgttgtttt tcaccgccgc gccttccgca ggcagaccaa
     4981 acgcgtccca gccgatcggc tgcaggacgt ttttgcccag catacgctgg tagcgggcga
     5041 tcacgtcacc gatggtgtag ttacgtacgt ggcccatgtg tagtcgacca gaaggatagg
     5101 ggaggataga caggcagtaa tacttctctt tgctctcgtc ttcggttact tcaaatgtgc
     5161 gcttctcatc ccaatgaagc tgtactttgg attctatctc ttccgggcgg tattgctctt
     5221 ccatggtggc ggcaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt
     5281 cgtattaatt tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctaga
     5341 ccaagtgacg atcacagcga tccacaaaca agaaccgcga cccaaatccc ggctgcgacg
     5401 gaactagctg tgccacaccc ggcgcgtcct tatataatca tcggcgttca ccgccccacg
     5461 gagatccctc cgcagaatcg ccgagaaggg actacttttc ctcgcctgtt ccgctctctg
     5521 gaaagaaaac cagtgcccta gagtcaccca agtcccgtcc taaaatgtcc ttctgctgat
     5581 actggggttc taaggccgag tcttatgagc agcgggccgc tgtcctgagc gtccgggcgg
     5641 aaggatcagg acgctcgctg cgcccttcgt ctgacgtggc agcgctcgcc gtgaggaggg
     5701 gggcgcccgc gggaggcgcc aaaacccggc gcggaggcct tcgaacggcc actagtcaat
     5761 aatcaatgtc aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acggatccaa
     5821 aaaatacccg gagcgggact tgaacccgca cagcgcgaac gccgagggat ttagaatccc
     5881 ttgtgtctac cgattccacc atccgggcgg tgtctctatc actgataggg aacttataag
     5941 tctctatcac tgatagggat ttcacgttta tggtgatttc ccagaacaca tagcgacatg
     6001 caaatattag atccaaaaaa tacccggagc gggacttgaa cccgcacagc gcgaacgccg
     6061 agggatttag aatcccttgt gtctaccgat tccaccatcc gggcggtgtc tctatcactg
     6121 atagggaact tataagtctc tatcactgat agggatttca cgtttatggt gatttcccag
     6181 aacacatagc gacatgcaaa tattagatcc aaaaaatacc cggagcggga cttgaacccg
     6241 cacagcgcga acgccgaggg atttagaatc ccttgtgtct accgattcca ccatccgggc
     6301 ggtgtctcta tcactgatag ggaacttata agtctctatc actgataggg atttcacgtt
     6361 tatggtgatt tcccagaaca catagcgaca tgcaaatatt agatccaaaa aatacccgga
     6421 gcgggacttg aacccgcaca gcgcgaacgc cgagggattt agaatccctt gtgtctaccg
     6481 attccaccat ccgggcggtg tctctatcac tgatagggaa cttataagtc tctatcactg
     6541 atagggattt cacgtttatg gtgatttccc agaacacata gcgacatgca aatattagat
     6601 ccaaaaaata cccggagcgg gacttgaacc cgcacagcgc gaacgccgag ggatttagaa
     6661 tcccttgtgt ctaccgattc caccatccgg gcggtgtctc tatcactgat agggaactta
     6721 taagtctcta tcactgatag ggatttcacg tttatggtga tttcccagaa cacatagcga
     6781 catgcaaata ttagatcctg caggctgatc actgcttgag cctagttatt aatagtaatc
     6841 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     6901 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     6961 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     7021 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     7081 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     7141 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
     7201 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
     7261 tattttttaa ttattttgtg cagcgatggg ggcggggggg ggggggggcg cgcgccaggs
     7321 ggggsggggs ggggsgaggg gsggggsggg gsgaggsgga gaggtgcggc ggcagccaat
     7381 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
     7441 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
     7501 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
     7561 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
     7621 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
     7681 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
     7741 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt
     7801 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
     7861 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
     7921 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
     7981 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
     8041 caggkggggg tgccgggcgg ggcggggccg ccycgggccg gggrgggctc gggggrgggg
     8101 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
     8161 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
     8221 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
     8281 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     8341 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     8401 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     8461 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     8521 tttggcaaag aattggccaa ggaggccacc atggtgagca agggcgagga gctgttcacc
     8581 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     8641 tccggcgagg gcgagggcga tgccacctag ggcaagctga ccctgaagtt catctgcacc
     8701 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     8761 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     8821 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     8881 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     8941 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     9001 gtctgaatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     9061 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     9121 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     9181 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     9241 actctcggca tggacgagct gtacaagggg cccttcgaac aaaaactcat ctcagaagag
     9301 gatctgaata tgcataccgg tcatcatcac catcaccatc atcaccatca ccatcattaa
     9361 ggcctctaag gccgaattca acgcgttaag tcgacaatca acctctggat tacaaaattt
     9421 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     9481 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     9541 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     9601 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     9661 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     9721 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     9781 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     9841 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     9901 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     9961 tctccctttg ggccgcctcc ccgcgtcgac tttaactcgg ccagcacagt ggtcgatcga
    10021 ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg
    10081 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
    10141 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
    10201 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg
    10261 ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg
    10321 tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt
    10381 ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat
    10441 ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctttgc ggccgcggtc
    10501 cgtatactcc ggaatattaa tagatcatgg agataattaa aatgataacc atctcgcaaa
    10561 taaataagta ttttactgtt ttcgtaacag ttttgtaata aaaaaaccta taaatattcc
    10621 ggattattca taccgtccca ccatcgggcg cgaactccta aaaaaccgcc accatgaagt
    10681 gccttttgta cttagccttt ttattcattg gggtgaattg caagttcacc atagtttttc
    10741 cacacaacca aaaaggaaac tggaaaaatg ttccttctaa ttaccattat tgcccgtcaa
    10801 gctcagattt aaattggcat aatgacttaa taggcacagc cttacaagtc aaaatgccca
    10861 agagtcacaa ggctattcaa gcagacggtt ggatgtgtca tgcttccaaa tgggtcacta
    10921 cttgtgattt ccgctggtat ggaccgaagt atataacaca ttccatccga tccttcactc
    10981 catctgtaga acaatgcaag gaaagcattg aacaaacgaa acaaggaact tggctgaatc
    11041 caggcttccc tcctcaaagt tgtggatatg caactgtgac ggatgccgaa gcagtgattg
    11101 tccaggtgac tcctcaccat gtgctggttg atgaatacac aggagaatgg gttgattcac
    11161 agttcatcaa cggaaaatgc agcaattaca tatgccccac tgtccataac tctacaacct
    11221 ggcattctga ctataaggtc aaagggctat gtgattctaa cctcatttcc atggacatca
    11281 ccttcttctc agaggacgga gagctatcat ccctgggaaa ggagggcaca gggttcagaa
    11341 gtaactactt tgcttatgaa actggaggca aggcctgcaa aatgcaatac tgcaagcatt
    11401 ggggagtcag actcccatca ggtgtctggt tcgagatggc tgataaggat ctctttgctg
    11461 cagccagatt ccctgaatgc ccagaagggt caagtatctc tgctccatct cagacctcag
    11521 tggatgtaag tctaattcag gacgttgaga ggatcttgga ttattccctc tgccaagaaa
    11581 cctggagcaa aatcagagcg ggtcttccaa tctctccagt ggatctcagc tatcttgctc
    11641 ctaaaaaccc aggaaccggt cctgctttca ccataatcaa tggtacccta aaatactttg
    11701 agaccagata catcagagtc gatattgctg ctccaatcct ctcaagaatg gtcggaatga
    11761 tcagtggaac taccacagaa agggaactgt gggatgactg ggcaccatat gaagacgtgg
    11821 aaattggacc caatggagtt ctgaggacca gttcaggata taagtttcct ttatacatga
    11881 ttggacatgg tatgttggac tccgatcttc atcttagctc aaaggctcag gtgttcgaac
    11941 atcctcacat tcaagacgct gcttcgcaac ttcctgatga tgagagttta ttttttggtg
    12001 atactgggct atccaaaaat ccaatcgagc ttgtagaagg ttggttcagt agttggaaaa
    12061 gctctattgc ctcttttttc tttatcatag ggttaatcat tggactattc ttggttctcc
    12121 gagttggtat ccatctttgc attaaattaa agcacaccaa gaaaagacag atttatacag
    12181 acatagagat gaaccgactt ggaaagtgat aaaagcttgt cgagaagtac tagaggatca
    12241 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
    12301 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
    12361 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
    12421 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatctgat
    12481 cactgcttga gcctaggaga tccgaaccag ataagtgaaa tctagttcca aactattttg
    12541 tcatttttaa ttttcgtatt agcttacgac gctacaccca gttcccatct attttgtcac
    12601 tcttccctaa ataatcctta aaaactccat ttccacccct cccagttccc aactattttg
    12661 tccgcccaca gcggggcatt tttcttcctg ttatgttttt aatcaaacat cctgccaact
    12721 ccatgtgaca aaccgtcatc ttcggctact ttttctctgt cacagaatga aaatttttct
    12781 gtcatctctt cgttattaat gtttgtaatt gactgaatat caacgcttat ttgcagcctg
    12841 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
    12901 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
    12961 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
    13021 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
    13081 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
    13141 ttcttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
    13201 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
    13261 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
    13321 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
    13381 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
    13441 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
    13501 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
    13561 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
    13621 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
    13681 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
    13741 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
    13801 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
    13861 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
    13921 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
    13981 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
    14041 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
    14101 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
    14161 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
    14221 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
    14281 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
    14341 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
    14401 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
    14461 aggatcttc
//
