LOCUS       sequence-272127-       13678 bp    DNA     circular SYN 14-APR-2025
DEFINITION  PiggyBac vector for establishement of doxycycline-inducible Sp dCas9
            expression cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13678)
  AUTHORS   Taghbalout A, Du M, Jillette N, Rosikiewicz W, Rath A, Heinen CD, Li
            S, Cheng AW
  TITLE     Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated 
            RNA-guided coupling of methylcytosine oxidation and DNA repair 
            pathways.
  JOURNAL   Nat Commun. 2019 Sep 20;10(1):4296. doi: 10.1038/s41467-019-12339-7.
  PUBMED    31541098
REFERENCE   2  (bases 1 to 13678)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-09-20"; volume: "10(1):4296. doi"; pages: " 
            10.1038/s41467-019-12339-7"
COMMENT     Sequence Label: pAT1089_PB-EF1a-Blast_2A_rtTA3-SV40pA_pCW-dCas9
FEATURES             Location/Qualifiers
     source          1..13678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   299..333
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1485..1566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1522..1541)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1576..1595
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1603..1611,1612..1647,1648..1650,1651..1686,
                     1687..1689,1690..1725,1726..1731,1732..2349))
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(2350..2403)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    complement(2862..2886)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2947..4125)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2956..3894
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2979..2999)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        4142..4456
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    4150..4168
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4186..4204
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4221..4239
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4257..4275
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4293..4311
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4328..4346
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4364..4382
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4429..4453
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4481..4505
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4532..4558
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4562..4582
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4592..4615,4616..4618,4619..7174,7175..7177,
                     7178..8692)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(4592..4615,4616..4618,4619..7105,7106..7108,
                     7109..8692)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8696..8716
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(8744..8768)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             8821..8862
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(8900..8917)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8906..9130
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   9950..10012
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10050..10069)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10051..10069)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10076..10093)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10076..10092)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10085..10107)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             10230..10532
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     10411..10430
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             10881..11675
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             10881..11675
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(10935..10954)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     11545..11564
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(11925..12716,12717..12785))
                     /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     12548..12567
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12909..13497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13398..13417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13651..13668
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttttaacc
      301 ctagaaagat agtctgcgta aaattgacgc atgcattctt gaaatattgc tctctctttc
      361 taaatagcgc gaatccgtcg ctgtgcattt aggacatctc agtcgccgct tggagctccc
      421 gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt gaactataac gaccgcgtga
      481 gtcaaaatga cgcatgatta tcttttacgt gacttttaag atttaactca tacgataatt
      541 atattgttat ttcatgttct acttacgtga taacttatta tatatatatt ttcttgttat
      601 agatatcaac tagaatgcta gacctttcgt cttcaagaat tccgatcata ttcaataacc
      661 cttaattagg tccctcgaag aggttcactg gcgcgttgga tccccgggta ccgagttggg
      721 agctcacggg gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc
      781 tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc
      841 cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc
      901 tctgaacgct tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag
      961 ctttaggctg aaagagagat ttagaatgac agtctagtgg gagctcacgg ggacagcccc
     1021 cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc
     1081 gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg
     1141 gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc
     1201 tctttgagcc tgcagacacc tggggggata cggggaaaaa gctttaggct gaaagagaga
     1261 tttagaatga cagaactcga tttcattgca gactggcgcg ccgccttttt acggttcctg
     1321 gccttttgct ggccttttgc tcacatgtca cgtgaggcct taacgtctcg ccctttggtc
     1381 tccccctctt aagtaccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     1441 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     1501 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1561 cactgcattc tagttgtggt ttgtccaaac tcatcggcta gcttacccgg ggagcatgtc
     1621 aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt cgtcaagggc
     1681 atcggctggg agcatgtcta agtcaaaatc gtcaagggcg tcggtcggcc cgccgctttc
     1741 gcactttagc tgtttctcca ggccacatat gattagttcc aggccgaaaa ggaaggcagg
     1801 ttcggctccc tgccggtcga acagctcaat tgcttgtctc agaagtgggg gcatagaatc
     1861 ggtggtaggt gtctctcttt cctcttttgc tacttgatgc tcctgttcct ccaatacgca
     1921 gcccagtgta aagtggccca cggcggacag agcgtacagt gcgttctcca gggagaagcc
     1981 ttgctgacac aggaacgcga gctgattttc cagggtttcg tactgtttct ctgttgggcg
     2041 ggtgccgaga tgcactttag ccccgtcgcg atgtgagagg agagcacagc ggtatgactt
     2101 ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc agggggcaga agtgggtatg
     2161 atgcctgtcc agcatctcga ttggcagggc atcgagcagg gcccgcttgt tcttcacgtg
     2221 ccagtacagg gtaggctgct caactcccag cttttgagcg agtttccttg tcgtcaggcc
     2281 ttcgataccg acaccattga gtaattccag agctccgttt atgactttgc tcttgtccag
     2341 tctagacatt ggaccagggt tttcttcaac atcaccacaa gtgaggagag aacctctacc
     2401 ttcggcaccg ggatttcggg tatatttgag tggaatgagt tcttcaatcg tagttttgac
     2461 taacttgcca ttcatttcta ttaacacaaa acaatctggt gcatagtctg aaatcaactc
     2521 cctacacata ccacaaggac ttaccactcg aatacttcta tctacttcgt cagaataagg
     2581 gtgtctaaca gctacaatcg tgtcaaaatc cttttgtcca ttcgaaactg cactaccaat
     2641 cgcaatggct tctgcacaaa cagttactcg tcctatatac gcttcaatat gtactgccga
     2701 aatgatttct cctgttttcg tacgaattgc cgctcccaca tgatgtttat tatcctcata
     2761 aagcattgta atcttctctg tcgctacttc tactaattct agatcctgtt gagaaatgtt
     2821 aaatgttttc atggtggcgg cactagtaag ggcgaattcg gagcctgctt ttttgtacaa
     2881 acttgttgat atctgcagaa ttccaccaca ctggactagt ggatccgagc tcggtaccaa
     2941 gcttcttcac gacacctgaa atggaagaaa aaaactttga accactgtct gaggcttgag
     3001 aatgaaccaa gatccaaact caaaaagggc aaattccaag gagaattaca tcaagtgcca
     3061 agctggccta acttcagtct ccacccactc agtgtgggga aactccatcg cataaaaccc
     3121 ctccccccaa cctaaagacg acgtactcca aaagctcgag aactaatcga ggtgcctgga
     3181 cggcgcccgg tactccgtgg agtcacatga agcgacggct gaggacggaa aggccctttt
     3241 cctttgtgtg ggtgactcac ccgcccgctc tcccgagcgc cgcgtcctcc attttgagct
     3301 ccctgcagca gggccgggaa gcggccatct ttccgctcac gcaactggtg ccgaccgggc
     3361 cagccttgcc gcccagggcg gggcgataca cggcggcgcg aggccaggca ccagagcagg
     3421 ccggccagct tgagactacc cccgtccgat tctcggtggc cgcgctcgca ggccccgcct
     3481 cgccgaacat gtgcgctggg acgcacgggc cccgtcgccg cccgcggccc caaaaaccga
     3541 aataccagtg tgcagatctt ggcccgcatt tacaagacta tcttgccaga aaaaaagcgt
     3601 cgcagcaggt catcaaaaat tttaaatggc tagagactta tcgaaagcag cgagacaggc
     3661 gcgaaggtgc caccagattc gcacgcggcg gccccagcgc ccaggccagg cctcaactca
     3721 agcacgaggc gaaggggctc cttaagcgca aggcctcgaa ctctcccacc cacttccaac
     3781 ccgaagctcg ggatcaagaa tcacgtactg cagccaggtg gaagtaattc aaggcacgca
     3841 agggccataa cccgtaaaga ggccaggccc gcgggaacca cacacggcac ttacctgtgt
     3901 tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac ttatatacgg
     3961 ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt cccagtttac
     4021 cccgcgccac cttctctagg caccggttca attgccgacc cctcccccca acttctcggg
     4081 gactgtgggc gatgtgcgct ctgcccactg acgggcaccg gagcctcacg atcgatatgt
     4141 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
     4201 gagaacgatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc
     4261 tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc
     4321 gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga
     4381 gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt
     4441 gaaccgtcag atcgcctgga gaattggcta ggcaccggtg acaagtttgt acaaaaaagc
     4501 aggctccgaa ttcaccggtg ccgccaccat gtacccatac gatgttccag attacgcttc
     4561 gccgaagaaa aagcgcaagg tcgaagcgtc cgacaagaag tacagcatcg gcctggccat
     4621 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     4681 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     4741 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     4801 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     4861 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     4921 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     4981 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     5041 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     5101 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     5161 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     5221 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     5281 cgagaagaag aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa
     5341 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     5401 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     5461 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     5521 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     5581 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     5641 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     5701 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     5761 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     5821 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     5881 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     5941 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     6001 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     6061 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     6121 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     6181 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     6241 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     6301 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     6361 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     6421 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     6481 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     6541 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     6601 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     6661 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     6721 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     6781 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     6841 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     6901 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     6961 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     7021 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     7081 ccggctgtcc gactacgatg tggacgccat cgtgcctcag agctttctga aggacgactc
     7141 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     7201 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     7261 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     7321 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     7381 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     7441 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     7501 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     7561 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     7621 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     7681 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     7741 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     7801 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     7861 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     7921 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     7981 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     8041 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     8101 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     8161 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     8221 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     8281 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     8341 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     8401 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     8461 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     8521 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     8581 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     8641 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acagccccaa
     8701 gaagaagaga aaggtggagg ccagctgatt aattaagaat tcgacccagc tttcttgtac
     8761 aaagtggttg atatccagca cagtggcggc cgctcgagtc tagagggccc gcggttcgaa
     8821 ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg ttaggggccc
     8881 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     8941 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     9001 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     9061 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     9121 ggctctatgg cgacgcgcct ggatccccgg gtaccgagtt gggagctcac ggggacagcc
     9181 cccccccaaa gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag
     9241 ccgcccgggg ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg
     9301 gggacagccc gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct
     9361 gctctttgag cctgcagaca cctgggggga tacggggaaa aagctttagg ctgaaagaga
     9421 gatttagaat gacagtctag tgggagctca cggggacagc ccccccccaa agcccccagg
     9481 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     9541 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     9601 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     9661 acctgggggg atacggggaa aaagctttag gctgaaagag agatttagaa tgacagaact
     9721 cgatttcatt gcagactggc cggccactag tacgcgccgg ctcgacatac tagttaaaag
     9781 ttttgttact ttatagaaga aattttgagt ttttgttttt ttttaataaa taaataaaca
     9841 taaataaatt gtttgttgaa tttattatta gtatgtaagt gtaaatataa taaaacttaa
     9901 tatctattca aattaataaa taaacctcga tatacagacc gataaaacac atgcgtcaat
     9961 tttacgcatg attatcttta acgtacgtca caatatgatt atctttctag ggttaaaagg
    10021 gcgaattcgc ggccgctaaa ttcaattcgc cctatagtga gtcgtattac aattcactgg
    10081 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
    10141 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
    10201 cccaacagtt gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga
    10261 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga
    10321 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc
    10381 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
    10441 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
    10501 acgccattaa cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga
    10561 tcttcaccta gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
    10621 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
    10681 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg
    10741 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
    10801 ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga
    10861 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
    10921 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
    10981 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
    11041 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
    11101 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
    11161 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
    11221 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
    11281 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
    11341 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
    11401 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
    11461 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
    11521 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
    11581 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
    11641 tcgccttcta tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga
    11701 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatcagg tggcactttt
    11761 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
    11821 ccgctcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
    11881 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
    11941 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
    12001 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
    12061 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
    12121 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
    12181 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
    12241 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
    12301 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
    12361 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
    12421 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
    12481 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
    12541 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
    12601 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
    12661 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
    12721 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
    12781 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgacc
    12841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
    12901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
    12961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
    13021 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
    13081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
    13141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
    13201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
    13261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
    13321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
    13381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
    13441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
    13501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
    13561 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
    13621 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
