LOCUS       sequence-378368-       14313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac integration and dox-inducible expression of 
            KRAB-dCas9-HA-tagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14313)
  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 14313)
  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-KRAB
FEATURES             Location/Qualifiers
     source          1..14313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     143..162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(381..485)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(511..966)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(648..669)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     860..879
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1093..1115
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1107..1124
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1108..1124
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1254..1316
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1754..1772
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1789..1807
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1825..1843
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1861..1879
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1896..1914
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1932..1950
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        1962..2000
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1997..2021
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2077..2086
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2113..2298
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             2320..6423
                     /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             6427..6453
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6433..6459
                     /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             6472..6492
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6499..6519
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6559..6615
                     /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             6616..7317
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    7340..7928
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(7811..7822)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7935..9146
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     8983..9002
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     protein_bind    complement(9159..9192)
                     /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             9199..9906
                     /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(9907..9940)
                     /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    9963..10535
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(10130..10147)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     10357..10376
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             10538..11137
                     /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     11037..11057
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    11263..11384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11300..11319)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11354..11373
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   12147..12181
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12672..12688)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12672..12688)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12685..12707)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12696..12712
                     /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(12720..12726,12727..12744,12745..12750))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12765..12786
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12903..12920)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13074..13662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13154..13173)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13833..14313,1..311,312..380))
                     /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 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
       61 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
      121 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
      181 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
      241 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
      301 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
      361 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
      421 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
      481 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
      541 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
      601 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
      661 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
      721 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
      781 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
      841 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
      901 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
      961 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     1021 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     1081 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcctcgttca
     1141 ttcacgtttt tgaacccgtg gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg
     1201 tgatggagca gatgaagatg ctcgacacgc tgcagaacac gcagctagat taaccctaga
     1261 aagataatca tattgtgacg tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt
     1321 ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag ttttattata
     1381 tttacactta catactaata ataaattcaa caaacaattt atttatgttt atttatttat
     1441 taaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta tgagggacag
     1501 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     1561 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     1621 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     1681 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaggcctcc acggccagac
     1741 tagacgagtt tactccctat cagtgataga gaacgatgtc gagtttactc cctatcagtg
     1801 atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac
     1861 tccctatcag tgatagagaa cgtatgtcga gtttatccct atcagtgata gagaacgtat
     1921 gtcgagttta ctccctatca gtgatagaga acgtatgtcg aggtaggcgt gtacggtggg
     1981 aggcctatat aagcagagct cgtttagtga accgtcagat cgcaccggtg gtaccgactc
     2041 gaattccgcc cgggctcgag cttaaggtta attaacgcca ccatggatgc taagtcacta
     2101 actgcctggt cccggacact ggtgaccttc aaggatgtat ttgtggactt caccagggag
     2161 gagtggaagc tgctggacac tgctcagcag atcgtgtaca gaaatgtgat gctggagaac
     2221 tataagaacc tggtttcctt gggttatcag cttactaagc cagatgtgat cctccggttg
     2281 gagaagggag aagagcccgg cggttccggc ggagggtcga tggacaagaa gtacagcatc
     2341 ggcctggcca tcggcaccaa ctctgtaggc tgggccgtga tcaccgacga gtacaaggtg
     2401 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     2461 atcggcgccc tgctgttcga cagcggagaa acagccgagg ccacccggct gaagagaacc
     2521 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     2581 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     2641 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     2701 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     2761 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     2821 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     2881 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccgg cggcgtggac
     2941 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     3001 cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc
     3061 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     3121 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     3181 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     3241 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     3301 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     3361 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacatcga tggcggagcc
     3421 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     3481 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     3541 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     3601 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     3661 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     3721 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc
     3781 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     3841 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg
     3901 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaagcc
     3961 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     4021 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     4081 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     4141 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     4201 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     4261 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     4321 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     4381 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     4441 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     4501 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     4561 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     4621 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     4681 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     4741 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     4801 ctggacatca accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg
     4861 aaggacgact ccatcgataa caaagtgctg actcggagcg acaagaaccg gggcaagagc
     4921 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg ccagctgctg
     4981 aatgccaagc tgattaccca gaggaagttc gacaatctga ccaaggccga gagaggcggc
     5041 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     5101 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaacgac
     5161 aaactgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     5221 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     5281 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     5341 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     5401 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     5461 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     5521 gaaacaggcg agatcgtgtg ggataagggc cgggactttg ccaccgtgcg gaaagtgctg
     5581 tctatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     5641 gagtctatcc tgcccaagag gaacagcgac aagctgatcg ccagaaagaa ggactgggac
     5701 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     5761 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     5821 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     5881 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     5941 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     6001 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     6061 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaaaca ctacctggac
     6121 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     6181 gacaaggtgc tgagcgccta caacaagcac agagacaagc ctatcagaga gcaggccgag
     6241 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     6301 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     6361 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     6421 gacgcctatc cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa
     6481 aaacgcaagg tggaagatcc taagaaaaag cggaaagtgg acggcattgg tagtgggagc
     6541 aacggcagca gcggatccgc tactaacttc agcctgctga agcaggctgg agacgtggag
     6601 gagaaccctg gacctatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg
     6661 gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac
     6721 gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc
     6781 gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc
     6841 atccccgact tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca
     6901 tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc
     6961 atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag
     7021 aaaacactcg gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa
     7081 ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag
     7141 accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg
     7201 gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag
     7261 gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct taattaatgt
     7321 acaaggcggc cgcgtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     7381 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     7441 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     7501 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     7561 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     7621 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     7681 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     7741 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     7801 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     7861 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     7921 ctccccgcac gcgtggcctc cgcgccgggt tttggcgcct cccgcgggcg cccccctcct
     7981 cacggcgagc gctgccacgt cagacgaagg gcgcagcgag cgtcctgatc cttccgcccg
     8041 gacgctcagg acagcggccc gctgctcata agactcggcc ttagaacccc agtatcagca
     8101 gaaggacatt ttaggacggg acttgggtga ctctagggca ctggttttct ttccagagag
     8161 cggaacaggc gaggaaaagt agtcccttct cggcgattct gcggagggat ctccgtgggg
     8221 cggtgaacgc cgatgattat ataaggacgc gccgggtgtg gcacagctag ttccgtcgca
     8281 gccgggattt gggtcgcggt tcttgtttgt ggatcgctgt gatcgtcact tggtgagtag
     8341 cgggctgctg ggctggccgg ggctttcgtg gccgccgggc cgctcggtgg gacggaagcg
     8401 tgtggagaga ccgccaaggg ctgtagtctg ggtccgcgag caaggttgcc ctgaactggg
     8461 ggttgggggg agcgcagcaa aatggcggct gttcccgagt cttgaatgga agacgcttgt
     8521 gaggcgggct gtgaggtcgt tgaaacaagg tggggggcat ggtgggcggc aagaacccaa
     8581 ggtcttgagg ccttcgctaa tgcgggaaag ctcttattcg ggtgagatgg gctggggcac
     8641 catctgggga ccctgacgtg aagtttgtca ctgactggag aactcggttt gtcgtctgtt
     8701 gcgggggcgg cagttatggc ggtgccgttg ggcagtgcac ccgtaccttt gggagcgcgc
     8761 gccctcgtcg tgtcgtgacg tcacccgttc tgttggctta taatgcaggg tggggccacc
     8821 tgccggtagg tgtgcggtag gcttttctcc gtcgcaggac gcagggttcg ggcctagggt
     8881 aggctctcct gaatcgacag gcgccggacc tctggtgagg ggagggataa gtgaggcgtc
     8941 agtttctttg gtcggtttta tgtacctatc ttcttaagta gctgaagctc cggttttgaa
     9001 ctatgcgctc ggggttggcg agtgtgtttt gtgaagtttt ttaggcacct tttgaaatgt
     9061 aatcatttgg gtcaatatgt aattttcagt gttagactag taaattgtcc gctaaattct
     9121 ggccgttttt ggcttttttg ttagacgcta gcggatccat aacttcgtat agtatacatt
     9181 atacgaagtt atgccaccat gtctaggctg gacaagagca aagtcataaa cggagctctg
     9241 gaattactca atggtgtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg
     9301 ggagttgagc agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc
     9361 ctgccaatcg agatgctgga caggcatcat acccacttct gccccctgga aggcgagtca
     9421 tggcaagact ttctgcggaa caacgccaag tcataccgct gtgctctcct ctcacatcgc
     9481 gacggggcta aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa
     9541 aatcagctcg cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg
     9601 tccgccgtgg gccactttac actgggctgc gtattggagg aacaggagca tcaagtagca
     9661 aaagaggaaa gagagacacc taccaccgat tctatgcccc cacttctgag acaagcaatt
     9721 gagctgttcg accggcaggg agccgaacct gccttccttt tcggcctgga actaatcata
     9781 tgtggcctgg agaaacagct aaagtgcgaa agcggcgggc cgaccgacgc ccttgacgat
     9841 tttgacttag acatgctccc agccgatgcc cttgacgatt ttgaccttga catgctcccc
     9901 gggtaaataa cttcgtatag tatacattat acgaagttat ggatccgcgg ccgcaaattc
     9961 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
    10021 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc
    10081 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
    10141 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
    10201 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
    10261 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
    10321 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
    10381 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
    10441 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
    10501 ggggacgtgg ttttcctttg aaaaacacga taataccatg gccaccgagt acaagcccac
    10561 ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt
    10621 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
    10681 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
    10741 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
    10801 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
    10861 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
    10921 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
    10981 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
    11041 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
    11101 accgcgcacc tggtgcatga cccgcaagcc cggtgccgtc gacaatcaac ctctggatta
    11161 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
    11221 atacgctgct ttaatgcctt tgtatcatgc gttaactaac taaacttgtt tattgcagct
    11281 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
    11341 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggaattga
    11401 ctcaaatgat gtcaattagt ctatcagaag ctcatctggt ctcccttccg ggggacaaga
    11461 catccctgtt taatatttaa acagcagtgt tcccaaactg ggttcttata tcccttgctc
    11521 tggtcaacca ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta aagaaacagt
    11581 ggcagccagg tttagccccg gaattgactg gattcctttt ttagggccca ttggtatggc
    11641 tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag
    11701 gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg
    11761 ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag
    11821 cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc ctgatatcta
    11881 taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata
    11941 attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact
    12001 cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg
    12061 ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag
    12121 aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg
    12181 gttaatctag ctgcatcagg atcatatcgt cgggtctttt ttccggctca gtcatcgccc
    12241 aagctggcgc tatctgggca tcggggagga agaagcccgt gccttttccc gcgaggttga
    12301 agcggcatgg aaagagtttg ccgaggatga ctgctgctgc attgacgttg agcgaaaacg
    12361 cacgtttacc atgatgattc gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt
    12421 cgttcaggcc acctgggata ccagttcgtc gcggcttttc cggacacagt tccggatggt
    12481 cagcccgaag cgcatcagca acccgaacaa taccggcgac agccggaact gccgtgccgg
    12541 tgtgcagatt aatgacagcg gtgcggcgct gggatattac gtcagcgagg acgggtatcc
    12601 tggctggatg ccgcagaaat ggacatggat accccgtgag ttacccggcg ggcgcgcttg
    12661 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
    12721 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
    12781 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
    12841 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
    12901 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
    12961 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
    13021 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
    13081 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
    13141 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
    13201 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
    13261 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
    13321 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
    13381 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
    13441 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
    13501 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
    13561 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
    13621 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
    13681 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
    13741 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
    13801 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
    13861 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
    13921 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
    13981 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
    14041 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
    14101 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
    14161 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
    14221 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
    14281 gtcagaagta agttggccgc agtgttatca ctc
//
