LOCUS       sequence-481082-       10972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows co-translational incorporation of the fluorescent ncAA Anap 
            directly into the target protein; expresses tRNACUA-Leu, AnapRS, and
            dominant negative ribosomal release factor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10972)
  AUTHORS   Obashi K, Strub MP, Taraska JW
  TITLE     A PIP2-stabilized syntaxin-1a structure mapped with transition metal
            ion FRET and unnatural fluorescent amino acids at the plasma 
            membrane.
  JOURNAL   Structure. 2025 Nov 24:S0969-2126(25)00388-0. doi: 
            10.1016/j.str.2025.10.001.
  PUBMED    41290003
REFERENCE   2  (bases 1 to 10972)
  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: "Structure. 
            2025 Nov 24:S0969-2126(25)00388-0. doi: 10.1016/j.str.2025.10.001."
COMMENT     Sequence Label: pANAP-P2A-eRF(E55D)
FEATURES             Location/Qualifiers
     source          1..10972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..113
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    67..85
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    95..113
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            join(118..151,152..154,155..201)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        215..309
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    263..281
                     /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"
     tRNA            join(314..347,348..350,351..397)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        411..505
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    459..477
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    487..505
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            join(510..543,544..546,547..593)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        607..701
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    655..673
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    683..701
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            join(706..739,740..742,743..789)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        803..897
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    851..869
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    879..897
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            join(902..935,936..938,939..985)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        999..1093
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    1047..1065
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1075..1093
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            join(1098..1131,1132..1134,1135..1181)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        1195..1289
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    1243..1261
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1271..1289
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            join(1294..1327,1328..1330,1331..1377)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        1391..1485
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     protein_bind    1439..1457
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1467..1485
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     tRNA            join(1490..1523,1524..1526,1527..1573)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     primer_bind     complement(1612..1631)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1803..2182
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2183..2386
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2337..2357
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2431..2450
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2431..2449
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2485..2494
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2491..5079
                     /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="MEEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCFSGPPYPS
                     GRLHMGHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPAEGAAVKNNTAPAPWTYDN
                     IAYMKNQLKMLGFGYDWSRELATCTPEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQ
                     TVLANEQVIDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTVKTMQRN
                     WIGRSEGVEITFNVNDYDNTLTVYTTRPDTFMGCTYLAVAAGHPLAQKAAENNPELAAF
                     IDECRNTKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPG
                     HDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAI
                     ADKLTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDV
                     VMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESSWVLARYTCPQYKEGMLDSEA
                     ANYWLPVDIAIGGIEHAIMESLYCRFFHKLMRDAGMVNSDEPVKQLLCQGMVLADAFYY
                     VGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVE
                     RYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKSDVAALNVDAL
                     TENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQDRALMQEA
                     LLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLVVVQVNGKVRA
                     KITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVVGGPV"
     CDS             5089..5145
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5146..5187
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             6496..6522
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(6563..6580)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6569..6793
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6839..7267
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6926..6945)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7136..7157
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7276..7296)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7281..7610
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7461..7596
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7523..7542
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             7677..8471
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7731..7750)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8341..8360
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    8645..8766
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8682..8701)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8736..8755
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8815..8831)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8815..8831)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8828..8850)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8839..8855
                     /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(8863..8869,8870..8887,8888..8893))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8908..8929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9046..9063)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9217..9805)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9297..9316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9976..10767,10768..10836))
                     /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     10599..10618
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10837..10941)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctaat atttgcatgt cgctatgtgt tctgggaaat caccataaac
       61 gtgaaatccc tatcagtgat agagacttat aagttcccta tcagtgatag agacaccgcc
      121 cggatggtgg aatcggtaga cacaagggat tctaaatccc tcggcgttcg cgctgtgcgg
      181 gttcaagtcc cgctccgggt attttttgga tctaatattt gcatgtcgct atgtgttctg
      241 ggaaatcacc ataaacgtga aatccctatc agtgatagag acttataagt tccctatcag
      301 tgatagagac accgcccgga tggtggaatc ggtagacaca agggattcta aatccctcgg
      361 cgttcgcgct gtgcgggttc aagtcccgct ccgggtattt tttggatcta atatttgcat
      421 gtcgctatgt gttctgggaa atcaccataa acgtgaaatc cctatcagtg atagagactt
      481 ataagttccc tatcagtgat agagacaccg cccggatggt ggaatcggta gacacaaggg
      541 attctaaatc cctcggcgtt cgcgctgtgc gggttcaagt cccgctccgg gtattttttg
      601 gatctaatat ttgcatgtcg ctatgtgttc tgggaaatca ccataaacgt gaaatcccta
      661 tcagtgatag agacttataa gttccctatc agtgatagag acaccgcccg gatggtggaa
      721 tcggtagaca caagggattc taaatccctc ggcgttcgcg ctgtgcgggt tcaagtcccg
      781 ctccgggtat tttttggatc taatatttgc atgtcgctat gtgttctggg aaatcaccat
      841 aaacgtgaaa tccctatcag tgatagagac ttataagttc cctatcagtg atagagacac
      901 cgcccggatg gtggaatcgg tagacacaag ggattctaaa tccctcggcg ttcgcgctgt
      961 gcgggttcaa gtcccgctcc gggtattttt tggatctaat atttgcatgt cgctatgtgt
     1021 tctgggaaat caccataaac gtgaaatccc tatcagtgat agagacttat aagttcccta
     1081 tcagtgatag agacaccgcc cggatggtgg aatcggtaga cacaagggat tctaaatccc
     1141 tcggcgttcg cgctgtgcgg gttcaagtcc cgctccgggt attttttgga tctaatattt
     1201 gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatccctatc agtgatagag
     1261 acttataagt tccctatcag tgatagagac accgcccgga tggtggaatc ggtagacaca
     1321 agggattcta aatccctcgg cgttcgcgct gtgcgggttc aagtcccgct ccgggtattt
     1381 tttggatcta atatttgcat gtcgctatgt gttctgggaa atcaccataa acgtgaaatc
     1441 cctatcagtg atagagactt ataagttccc tatcagtgat agagacaccg cccggatggt
     1501 ggaatcggta gacacaaggg attctaaatc cctcggcgtt cgcgctgtgc gggttcaagt
     1561 cccgctccgg gtattttttg gatctcccga tcccctatgg tgcactctca gtacaatctg
     1621 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     1681 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     1741 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     1801 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     1861 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     1921 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     1981 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2041 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2101 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2161 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     2221 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     2281 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     2341 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
     2401 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
     2461 tggctagcgt ttaaacttaa gcttgccacc atggaagagc aataccgccc ggaagagata
     2521 gaatccaaag tacagcttca ttgggatgag aagcgcacat ttgaagtaac cgaagacgag
     2581 agcaaagaga agtattactg cttttctggc cctccctatc cttctggtcg actacacatg
     2641 ggccacgtac gtaactacac catcggtgac gtgatcgccc gctaccagcg tatgctgggc
     2701 aaaaacgtcc tgcagccgat cggctgggac gcgtttggtc tgcctgcgga aggcgcggcg
     2761 gtgaaaaaca acaccgctcc ggcaccgtgg acgtacgaca acatcgcgta tatgaaaaac
     2821 cagctcaaaa tgctgggctt tggttatgac tggagccgcg agctggcaac ctgtacgccg
     2881 gaatactacc gttgggaaca gaaattcttc accgagctgt ataaaaaagg cctggtatat
     2941 aagaagactt ctgcggtcaa ctggtgtccg aacgaccaga ccgtactggc gaacgaacaa
     3001 gttatcgacg gctgctgctg gcgctgcgat accaaagttg aacgtaaaga gatcccgcag
     3061 tggtttatca aaatcactgc ttacgctgac gagctgctca acgatctgga taaactggat
     3121 cactggcctg acaccgttaa aaccatgcag cgtaactgga tcggtcgttc cgaaggcgtg
     3181 gagatcacct tcaacgttaa cgactatgac aacacgctga ccgtttacac tacccgcccg
     3241 gacaccttta tgggttgtac ctacctggcg gtagctgcgg gtcatccgct ggcgcagaaa
     3301 gcggcggaaa ataatcctga actggcggcc tttattgacg aatgccgtaa caccaaagtt
     3361 gccgaagctg aaatggcgac gatggagaaa aaaggcgtcg atactggctt taaagcggtt
     3421 cacccattaa cgggcgaaga aattcccgtt tgggcagcaa acttcgtatt gatggagtac
     3481 ggcacgggcg cagttatggc ggtaccgggg cacgaccagc gcgactacga gtttgcctct
     3541 aaatacggcc tgaacatcaa accggttatc ctggcagctg acggctctga gccagatctt
     3601 tctcagcaag ccctgactga aaaaggcgtg ctgttcaact ctggcgagtt caacggtctt
     3661 gaccatgaag cggccttcaa cgccatcgcc gataaactga ctgcgatggg cgttggcgag
     3721 cgtaaagtga actaccgcct gcgcgactgg ggtgtttccc gtcagcgtta ctggggcgcg
     3781 ccgattccga tggtgactct agaagacggt accgtaatgc cgaccccgga cgaccagctg
     3841 ccggtgatcc tgccggagga tgtggtaatg gacggcatta ccagcccgat taaagcagat
     3901 ccggagtggg cgaaaactac cgttaacggt atgccagcac tgcgtgaaac cgacactttc
     3961 gacaccttta tggagtcctc ctgggttctt gcgcgctaca cttgcccgca gtacaaagaa
     4021 ggtatgctgg attccgaagc ggctaactac tggctgccgg tggatatcgc gattggtggt
     4081 attgaacacg ccattatgga gagtctctac tgtcgcttct tccacaaact gatgcgtgat
     4141 gcaggcatgg tgaactctga cgaaccagtt aaacagttgc tgtgtcaggg tatggtgctg
     4201 gcagatgcct tctactatgt tggcgaaaac ggcgaacgta actgggtttc cccggttgat
     4261 gctatcgttg aacgtgacga gaaaggccgt atcgtgaaag cgaaagatgc ggcaggccat
     4321 gaactggttt ataccggcat gagcaaaatg tccaagtcga agaacaacgg tatcgacccg
     4381 caggtgatgg ttgaacgtta cggcgcggac accgttcgtc tgtttatgat gtttgcttct
     4441 ccggctgata tgactctcga atggcaggaa tccggtgtgg aaggggctaa ccgcttcctg
     4501 aaacgtgtct ggaaactggt ttacgagcac acagcaaaaa gtgatgttgc ggcactgaac
     4561 gttgatgcgc tgactgaaaa tcagaaagcg ctgcgtcgcg atgtgcataa aacgatcgct
     4621 aaagtgaccg atgatatcgg ccgtcgtcag accttcaaca ccgcaattgc ggcgattatg
     4681 gagctgatga acaaactggc gaaagcacca accgatggcg agcaggatcg cgctctgatg
     4741 caggaagcac tgctggccgt tgtccgtatg cttaacccgt tcaccccgca catctgcttc
     4801 acgctgtggc aggaactgaa aggcgaaggc gatatcgaca acgcgccgtg gccggttgct
     4861 gacgaaaaag cgatggtgga agactccacg ctggtcgtgg tgcaggttaa cggtaaagtc
     4921 cgtgccaaaa tcaccgttcc ggtggacgca acggaagaac aggttcgcga acgtgctggc
     4981 caggaacatc tggtagcaaa atatcttgat ggcgttactg tacgtaaagt gatttacgta
     5041 ccaggtaaac tcctcaatct ggtcgttggc gggcccgttg gaagcggagc tactaacttc
     5101 agcctgctga agcaggctgg cgacgtggag gagaaccctg gacctggcaa gcctattccc
     5161 aaccccctgc tgggcctgga tagcaccgct gacgacccct ccgctgccga tagaaacgtg
     5221 gaaatctgga agatcaagaa actgattaag agcctggagg ctgccagagg aaacggcacc
     5281 agcatgatct ccctgattat ccctcccaag gatcagatca gcagggtcgc taagatgctc
     5341 gccgatgatt tcggcaccgc cagcaatatc aagagcaggg tgaacagact gagcgtgctg
     5401 ggcgccatta ccagcgtgca gcagagactg aagctgtaca acaaggtgcc ccccaacgga
     5461 ctggtggtgt actgtggaac catcgtgacc gaagagggca aggaaaaaaa agtgaacatc
     5521 gacttcgagc ccttcaaacc catcaacacc tccctctacc tgtgcgacaa taagttccac
     5581 accgaggccc tgacagctct gctcagcgac gactccaagt tcggcttcat tgtgatcgac
     5641 ggaagcggcg ctctgttcgg aaccctgcag ggcaacacca gggaggtgct ccacaagttc
     5701 accgtggacc tgcctaaaaa gcacggcaga ggcggccaga gcgctctgag attcgccagg
     5761 ctgagaatgg agaagaggca caactacgtg agaaaggtgg ccgaaaccgc cgtgcagctg
     5821 ttcattagcg gcgacaaggt gaacgtggcc ggcctggtgc tggccggctc cgccgacttt
     5881 aagaccgagc tgtcccagag cgatatgttc gatcagaggc tgcaaagcaa ggtgctgaag
     5941 ctggtggaca tcagctacgg aggcgagaac ggcttcaacc aggccatcga actcagcacc
     6001 gaggtgctga gcaacgtgaa attcattcaa gagaagaagc tcatcggcag gtacttcgac
     6061 gagatcagcc aggataccgg caagtactgc tttggagtgg aggataccct gaaggccctg
     6121 gagatgggcg ccgtggagat cctgattgtg tacgagaatc tggacatcat gagatatgtg
     6181 ctccactgcc agggcaccga ggaggagaaa atcctgtacc tcacccccga gcaggagaag
     6241 gacaaaagcc acttcaccga taaggagacc ggccaggaac acgagctgat tgagtccatg
     6301 cccctgctgg agtggtttgc caacaactac aagaaattcg gcgccaccct cgagatcgtc
     6361 accgacaagt cccaggaagg ctcccagttc gtcaagggct tcggcggcat cggaggcatc
     6421 ctcaggtaca gggtcgactt ccagggcatg gagtatcagg gcggcgacga cgagttcttc
     6481 gatctggacg actactaccc atacgatgtt ccagattacg cttaactcga gtctagaggg
     6541 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     6601 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     6661 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     6721 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     6781 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     6841 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     6901 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     6961 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     7021 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     7081 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     7141 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     7201 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     7261 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     7321 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     7381 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     7441 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     7501 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     7561 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     7621 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     7681 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     7741 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     7801 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     7861 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     7921 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     7981 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     8041 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     8101 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     8161 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     8221 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     8281 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     8341 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     8401 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     8461 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     8521 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     8581 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     8641 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     8701 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     8761 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     8821 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     8881 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     8941 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     9001 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     9061 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     9121 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     9181 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     9241 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     9301 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     9361 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     9421 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     9481 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     9541 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     9601 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     9661 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     9721 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     9781 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     9841 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     9901 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     9961 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
    10021 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
    10081 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
    10141 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
    10201 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
    10261 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
    10321 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
    10381 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
    10441 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
    10501 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
    10561 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
    10621 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
    10681 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
    10741 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
    10801 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
    10861 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
    10921 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
