LOCUS       sequence-378348-       15642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac integration and dox-inducible expression of 
            VPR-dCas9-HA-tagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15642)
  AUTHORS   Fuentes DR, Swigut T, Wysocka J
  TITLE     Systematic perturbation of retroviral LTRs reveals widespread 
            long-range effects on human gene regulation.
  JOURNAL   Elife. 2018 Aug 2;7. pii: 35989. doi: 10.7554/eLife.35989.
  PUBMED    30070637
REFERENCE   2  (bases 1 to 15642)
  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: "Elife."; 
            date: "2018-08-2"; volume: "7. pii: 35989. doi"; pages: " 
            10.7554/eLife.35989"
COMMENT     Sequence Label: PiggyBac dCas9-VPR
FEATURES             Location/Qualifiers
     source          1..15642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(249..353)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(379..834)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(516..537)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     728..747
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     961..983
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     975..992
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     976..992
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1122..1184
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1622..1640
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1657..1675
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1693..1711
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1729..1747
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1764..1782
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1800..1818
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        1830..1868
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1865..1889
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1927..2076
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             2101..2121
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2551..2907
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             2926..3495
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             3517..7620
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAGGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7624..7650
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7630..7656
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             7669..7689
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7696..7716
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7756..7812
                     /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             7813..8514
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    8537..9125
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(9008..9019)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        9132..10343
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     10180..10199
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     protein_bind    complement(10356..10389)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     CDS             10396..11103
                     /codon_start=1
                     /product="reverse tetracycline transactivator 3"
                     /label=rtTA3
                     /note="activates expression from Tet-responsive promoters 
                     in the presence of doxycycline"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPG"
     protein_bind    complement(11104..11137)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     misc_feature    11160..11732
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(11327..11344)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     11554..11573
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             11735..12334
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MATEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     12234..12254
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    12460..12581
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12497..12516)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12551..12570
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   13344..13378
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(13869..13885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13869..13885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13882..13904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13893..13909
                     /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(13917..13923,13924..13941,13942..13947))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13962..13983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14100..14117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14271..14859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14351..14370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15030..15642,1..179,180..248))
                     /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"
ORIGIN
        1 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
       61 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      121 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      181 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      241 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      301 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      361 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
      421 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
      481 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
      541 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
      601 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
      661 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
      721 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
      781 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
      841 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
      901 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
      961 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc ctcgttcatt cacgtttttg
     1021 aacccgtgga ggacgggcag actcgcggtg caaatgtgtt ttacagcgtg atggagcaga
     1081 tgaagatgct cgacacgctg cagaacacgc agctagatta accctagaaa gataatcata
     1141 ttgtgacgta cgttaaagat aatcatgtgt aaaattgacg catgtgtttt atcggtctgt
     1201 atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca
     1261 tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaacaa
     1321 aaactcaaaa tttcttctat aaagtaacaa aacttttatg agggacagcc cccccccaaa
     1381 gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg
     1441 ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc
     1501 gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag
     1561 cctgcagaca cctgggggga tacggggaaa aggcctccac ggccagacta gacgagttta
     1621 ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat agagaacgta
     1681 tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg
     1741 atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt cgagtttact
     1801 ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag gcctatataa
     1861 gcagagctcg tttagtgaac cgtcagatcg caccggtggt accgactcga attccgcccg
     1921 ggcatggacg cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat
     1981 tttgaccttg acatgcttgg ttcggatgcc cttgatgact ttgacctcga catgctcggc
     2041 agtgacgccc ttgatgattt cgacctggac atgctgatta actctagaag ttccggatct
     2101 ccgaaaaaga aacgcaaagt tggtagccag tacctgcccg acaccgacga ccggcaccgg
     2161 atcgaggaaa agcggaagcg gacctacgag acattcaaga gcatcatgaa gaagtccccc
     2221 ttcagcggcc ccaccgaccc tagacctcca cctagaagaa tcgccgtgcc cagcagatcc
     2281 agcgccagcg tgccaaaacc tgccccccag ccttacccct tcaccagcag cctgagcacc
     2341 atcaactacg acgagttccc taccatggtg ttccccagcg gccagatctc tcaggcctct
     2401 gctctggctc cagcccctcc tcaggtgctg cctcaggctc ctgctcctgc accagctcca
     2461 gccatggtgt ctgcactggc tcaggcacca gcacccgtgc ctgtgctggc tcctggacct
     2521 ccacaggctg tggctccacc agcccctaaa cctacacagg ccggcgaggg cacactgtct
     2581 gaagctctgc tgcagctgca gttcgacgac gaggatctgg gagccctgct gggaaacagc
     2641 accgatcctg ccgtgttcac cgacctggcc agcgtggaca acagcgagtt ccagcagctg
     2701 ctgaaccagg gcatccctgt ggcccctcac accaccgagc ccatgctgat ggaatacccc
     2761 gaggccatca cccggctcgt gacaggcgct cagaggcctc ctgatccagc tcctgcccct
     2821 ctgggagcac caggcctgcc taatggactg ctgtctggcg acgaggactt cagctctatc
     2881 gccgatatgg atttctcagc cttgctgggc tctggcagcg gcagccggga ttccagggaa
     2941 gggatgtttt tgccgaagcc tgaggccggc tccgctatta gtgacgtgtt tgagggccgc
     3001 gaggtgtgcc agccaaaacg aatccggcca tttcatcctc caggaagtcc atgggccaac
     3061 cgcccactcc ccgccagcct cgcaccaaca ccaaccggtc cagtacatga gccagtcggg
     3121 tcactgaccc cggcaccagt ccctcagcca ctggatccag cgcccgcagt gactcccgag
     3181 gccagtcacc tgttggagga tcccgatgaa gagacgagcc aggctgtcaa agcccttcgg
     3241 gagatggccg atactgtgat tccccagaag gaagaggctg caatctgtgg ccaaatggac
     3301 ctttcccatc cgcccccaag gggccatctg gatgagctga caaccacact tgagtccatg
     3361 accgaggatc tgaacctgga ctcacccctg accccggaat tgaacgagat tctggatacc
     3421 ttcctgaacg acgagtgcct cttgcatgcc atgcatatca gcacaggact gtccatcttc
     3481 gacacatctc tgtttggcgg ttccggcgga gggtcgatgg acaagaagta cagcatcggc
     3541 ctggccatcg gcaccaactc tgtaggctgg gccgtgatca ccgacgagta caaggtgccc
     3601 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     3661 ggcgccctgc tgttcgacag cggagaaaca gccgaggcca cccggctgaa gagaaccgcc
     3721 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     3781 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     3841 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     3901 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     3961 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     4021 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     4081 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccggcgg cgtggacgcc
     4141 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     4201 ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg
     4261 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     4321 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     4381 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     4441 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     4501 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     4561 gagattttct tcgaccagag caagaacggc tacgccggct acatcgatgg cggagccagc
     4621 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     4681 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     4741 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     4801 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     4861 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     4921 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgccagcgcc
     4981 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     5041 cccaagcaca gcctgctgta cgagtacttc accgtgtaca acgagctgac caaagtgaaa
     5101 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaagccatc
     5161 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     5221 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
     5281 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
     5341 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
     5401 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
     5461 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
     5521 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
     5581 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
     5641 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
     5701 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
     5761 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
     5821 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
     5881 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
     5941 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
     6001 gacatcaacc ggctgtccga ctacgatgtg gacgctatcg tgcctcagag ctttctgaag
     6061 gacgactcca tcgataacaa agtgctgact cggagcgaca agaaccgggg caagagcgac
     6121 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcgcca gctgctgaat
     6181 gccaagctga ttacccagag gaagttcgac aatctgacca aggccgagag aggcggcctg
     6241 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
     6301 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaacgacaaa
     6361 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
     6421 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
     6481 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
     6541 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
     6601 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
     6661 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
     6721 acaggcgaga tcgtgtggga taagggccgg gactttgcca ccgtgcggaa agtgctgtct
     6781 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
     6841 tctatcctgc ccaagaggaa cagcgacaag ctgatcgcca gaaagaagga ctgggaccct
     6901 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
     6961 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
     7021 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
     7081 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
     7141 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
     7201 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
     7261 cccgaggata atgagcagaa acagctgttt gtggaacagc acaaacacta cctggacgag
     7321 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
     7381 aaggtgctga gcgcctacaa caagcacaga gacaagccta tcagagagca ggccgagaat
     7441 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
     7501 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
     7561 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac
     7621 gcctatccct atgacgtgcc cgattatgcc agcctgggca gcggctcccc caagaaaaaa
     7681 cgcaaggtgg aagatcctaa gaaaaagcgg aaagtggacg gcattggtag tgggagcaac
     7741 ggcagcagcg gatccgctac taacttcagc ctgctgaagc aggctggaga cgtggaggag
     7801 aaccctggac ctatgagcga gctgattaag gagaacatgc acatgaagct gtacatggag
     7861 ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
     7921 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac
     7981 atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc
     8041 cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
     8101 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
     8161 tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa
     8221 acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc
     8281 agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc
     8341 acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac
     8401 tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg
     8461 gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa ttaatgtaca
     8521 aggcggccgc gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     8581 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     8641 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     8701 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     8761 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     8821 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     8881 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc
     8941 ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     9001 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     9061 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     9121 cccgcacgcg tggcctccgc gccgggtttt ggcgcctccc gcgggcgccc ccctcctcac
     9181 ggcgagcgct gccacgtcag acgaagggcg cagcgagcgt cctgatcctt ccgcccggac
     9241 gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa
     9301 ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg
     9361 aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg
     9421 tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc
     9481 gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg
     9541 gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt
     9601 ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt
     9661 tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag
     9721 gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt
     9781 cttgaggcct tcgctaatgc gggaaagctc ttattcgggt gagatgggct ggggcaccat
     9841 ctggggaccc tgacgtgaag tttgtcactg actggagaac tcggtttgtc gtctgttgcg
     9901 ggggcggcag ttatggcggt gccgttgggc agtgcacccg tacctttggg agcgcgcgcc
     9961 ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa tgcagggtgg ggccacctgc
    10021 cggtaggtgt gcggtaggct tttctccgtc gcaggacgca gggttcgggc ctagggtagg
    10081 ctctcctgaa tcgacaggcg ccggacctct ggtgagggga gggataagtg aggcgtcagt
    10141 ttctttggtc ggttttatgt acctatcttc ttaagtagct gaagctccgg ttttgaacta
    10201 tgcgctcggg gttggcgagt gtgttttgtg aagtttttta ggcacctttt gaaatgtaat
    10261 catttgggtc aatatgtaat tttcagtgtt agactagtaa attgtccgct aaattctggc
    10321 cgtttttggc ttttttgtta gacgctagcg gatccataac ttcgtatagt atacattata
    10381 cgaagttatg ccaccatgtc taggctggac aagagcaaag tcataaacgg agctctggaa
    10441 ttactcaatg gtgtcggtat cgaaggcctg acgacaagga aactcgctca aaagctggga
    10501 gttgagcagc ctaccctgta ctggcacgtg aagaacaagc gggccctgct cgatgccctg
    10561 ccaatcgaga tgctggacag gcatcatacc cacttctgcc ccctggaagg cgagtcatgg
    10621 caagactttc tgcggaacaa cgccaagtca taccgctgtg ctctcctctc acatcgcgac
    10681 ggggctaaag tgcatctcgg cacccgccca acagagaaac agtacgaaac cctggaaaat
    10741 cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga acgcactgta cgctctgtcc
    10801 gccgtgggcc actttacact gggctgcgta ttggaggaac aggagcatca agtagcaaaa
    10861 gaggaaagag agacacctac caccgattct atgcccccac ttctgagaca agcaattgag
    10921 ctgttcgacc ggcagggagc cgaacctgcc ttccttttcg gcctggaact aatcatatgt
    10981 ggcctggaga aacagctaaa gtgcgaaagc ggcgggccga ccgacgccct tgacgatttt
    11041 gacttagaca tgctcccagc cgatgccctt gacgattttg accttgacat gctccccggg
    11101 taaataactt cgtatagtat acattatacg aagttatgga tccgcggccg caaattccgc
    11161 ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
    11221 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
    11281 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
    11341 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
    11401 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
    11461 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
    11521 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
    11581 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
    11641 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
    11701 gacgtggttt tcctttgaaa aacacgataa taccatggcc accgagtaca agcccacggt
    11761 gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc
    11821 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
    11881 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
    11941 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
    12001 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
    12061 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
    12121 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
    12181 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
    12241 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
    12301 gcgcacctgg tgcatgaccc gcaagcccgg tgccgtcgac aatcaacctc tggattacaa
    12361 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
    12421 cgctgcttta atgcctttgt atcatgcgtt aactaactaa acttgtttat tgcagcttat
    12481 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
    12541 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gaattgactc
    12601 aaatgatgtc aattagtcta tcagaagctc atctggtctc ccttccgggg gacaagacat
    12661 ccctgtttaa tatttaaaca gcagtgttcc caaactgggt tcttatatcc cttgctctgg
    12721 tcaaccaggt tgcagggttt cctgtcctca caggaacgaa gtccctaaag aaacagtggc
    12781 agccaggttt agccccggaa ttgactggat tcctttttta gggcccattg gtatggcttt
    12841 ttccccgtat ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa
    12901 agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga
    12961 tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg
    13021 gggagggacg taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa
    13081 caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt
    13141 atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac
    13201 gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga
    13261 gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa
    13321 gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt
    13381 aatctagctg catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag
    13441 ctggcgctat ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc
    13501 ggcatggaaa gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac
    13561 gtttaccatg atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt
    13621 tcaggccacc tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag
    13681 cccgaagcgc atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt
    13741 gcagattaat gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg
    13801 ctggatgccg cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg
    13861 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
    13921 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
    13981 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
    14041 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
    14101 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
    14161 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
    14221 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
    14281 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
    14341 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
    14401 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
    14461 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
    14521 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
    14581 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
    14641 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
    14701 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
    14761 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
    14821 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
    14881 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
    14941 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
    15001 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
    15061 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
    15121 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
    15181 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
    15241 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
    15301 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
    15361 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
    15421 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
    15481 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
    15541 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
    15601 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cg
//
