LOCUS       sequence-451639-       14697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac integration and dox-inducible expression of VPR-dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14697)
  AUTHORS   Jensen CL, Chen LF, Swigut T, Crocker OJ, Yao D, Bassik MC, Ferrell 
            JE Jr, Boettiger AN, Wysocka J
  TITLE     Long-range regulation of transcription scales with genomic distance 
            in a gene-specific manner.
  JOURNAL   Mol Cell. 2025 Jan 16;85(2):347-361.e7. doi: 
            10.1016/j.molcel.2024.10.021. Epub 2024 Dec 2.
  PUBMED    39626660
REFERENCE   2  (bases 1 to 14697)
  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"; doi: 
            "10.1016/j.molcel.2024.10.021"; journalName: "Mol Cell"; date: 
            "2025-01-16- 16"; volume: "85"; issue: "2"; pages: "347-361.e7"
COMMENT     Sequence Label: pB-tre-dCas9-VPR
FEATURES             Location/Qualifiers
     source          1..14697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..27
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(246..350)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(376..831)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(513..534)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     725..744
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     958..980
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     972..989
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     973..989
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1119..1181
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1619..1637
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1654..1672
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1690..1708
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1726..1744
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1761..1779
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1797..1815
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        1827..1865
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1862..1886
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1924..2073
                     /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             2098..2118
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2548..2904
                     /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             2923..3492
                     /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             3514..7617
                     /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             7621..7647
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7627..7653
                     /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             7666..7686
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7693..7713
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    7793..8381
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(8264..8275)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        8388..9599
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     9436..9455
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     protein_bind    complement(9612..9645)
                     /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             9652..10359
                     /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(10360..10393)
                     /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    10416..10988
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(10583..10600)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     10810..10829
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             10991..11386
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    11512..11633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11549..11568)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11603..11622
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   12396..12430
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12921..12937)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12921..12937)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12934..12956)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12945..12961
                     /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(12969..12975,12976..12993,12994..12999))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13014..13035
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13152..13169)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13323..13911)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13403..13422)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14082..14697,1..176,177..245))
                     /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 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
       61 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
      121 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
      181 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
      241 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
      301 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
      361 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
      421 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
      481 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
      541 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
      601 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
      661 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
      721 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
      781 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
      841 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
      901 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
      961 agtcacgacg ttgtaaaacg acggccagtg agcgcgcctc gttcattcac gtttttgaac
     1021 ccgtggagga cgggcagact cgcggtgcaa atgtgtttta cagcgtgatg gagcagatga
     1081 agatgctcga cacgctgcag aacacgcagc tagattaacc ctagaaagat aatcatattg
     1141 tgacgtacgt taaagataat catgtgtaaa attgacgcat gtgttttatc ggtctgtata
     1201 tcgaggttta tttattaatt tgaatagata ttaagtttta ttatatttac acttacatac
     1261 taataataaa ttcaacaaac aatttattta tgtttattta tttattaaaa aaaacaaaaa
     1321 ctcaaaattt cttctataaa gtaacaaaac ttttatgagg gacagccccc ccccaaagcc
     1381 cccagggatg taattacgtc cctcccccgc tagggggcag cagcgagccg cccggggctc
     1441 cgctccggtc cggcgctccc cccgcatccc cgagccggca gcgtgcgggg acagcccggg
     1501 cacggggaag gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct
     1561 gcagacacct ggggggatac ggggaaaagg cctccacggc cagactagac gagtttactc
     1621 cctatcagtg atagagaacg atgtcgagtt tactccctat cagtgataga gaacgtatgt
     1681 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
     1741 gagaacgtat gtcgagttta tccctatcag tgatagagaa cgtatgtcga gtttactccc
     1801 tatcagtgat agagaacgta tgtcgaggta ggcgtgtacg gtgggaggcc tatataagca
     1861 gagctcgttt agtgaaccgt cagatcgcac cggtggtacc gactcgaatt ccgcccgggc
     1921 atggacgcat tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt
     1981 gaccttgaca tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt
     2041 gacgcccttg atgatttcga cctggacatg ctgattaact ctagaagttc cggatctccg
     2101 aaaaagaaac gcaaagttgg tagccagtac ctgcccgaca ccgacgaccg gcaccggatc
     2161 gaggaaaagc ggaagcggac ctacgagaca ttcaagagca tcatgaagaa gtcccccttc
     2221 agcggcccca ccgaccctag acctccacct agaagaatcg ccgtgcccag cagatccagc
     2281 gccagcgtgc caaaacctgc cccccagcct taccccttca ccagcagcct gagcaccatc
     2341 aactacgacg agttccctac catggtgttc cccagcggcc agatctctca ggcctctgct
     2401 ctggctccag cccctcctca ggtgctgcct caggctcctg ctcctgcacc agctccagcc
     2461 atggtgtctg cactggctca ggcaccagca cccgtgcctg tgctggctcc tggacctcca
     2521 caggctgtgg ctccaccagc ccctaaacct acacaggccg gcgagggcac actgtctgaa
     2581 gctctgctgc agctgcagtt cgacgacgag gatctgggag ccctgctggg aaacagcacc
     2641 gatcctgccg tgttcaccga cctggccagc gtggacaaca gcgagttcca gcagctgctg
     2701 aaccagggca tccctgtggc ccctcacacc accgagccca tgctgatgga ataccccgag
     2761 gccatcaccc ggctcgtgac aggcgctcag aggcctcctg atccagctcc tgcccctctg
     2821 ggagcaccag gcctgcctaa tggactgctg tctggcgacg aggacttcag ctctatcgcc
     2881 gatatggatt tctcagcctt gctgggctct ggcagcggca gccgggattc cagggaaggg
     2941 atgtttttgc cgaagcctga ggccggctcc gctattagtg acgtgtttga gggccgcgag
     3001 gtgtgccagc caaaacgaat ccggccattt catcctccag gaagtccatg ggccaaccgc
     3061 ccactccccg ccagcctcgc accaacacca accggtccag tacatgagcc agtcgggtca
     3121 ctgaccccgg caccagtccc tcagccactg gatccagcgc ccgcagtgac tcccgaggcc
     3181 agtcacctgt tggaggatcc cgatgaagag acgagccagg ctgtcaaagc ccttcgggag
     3241 atggccgata ctgtgattcc ccagaaggaa gaggctgcaa tctgtggcca aatggacctt
     3301 tcccatccgc ccccaagggg ccatctggat gagctgacaa ccacacttga gtccatgacc
     3361 gaggatctga acctggactc acccctgacc ccggaattga acgagattct ggataccttc
     3421 ctgaacgacg agtgcctctt gcatgccatg catatcagca caggactgtc catcttcgac
     3481 acatctctgt ttggcggttc cggcggaggg tcgatggaca agaagtacag catcggcctg
     3541 gccatcggca ccaactctgt aggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
     3601 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcggc
     3661 gccctgctgt tcgacagcgg agaaacagcc gaggccaccc ggctgaagag aaccgccaga
     3721 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
     3781 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
     3841 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
     3901 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
     3961 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
     4021 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
     4081 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccggcggcgt ggacgccaag
     4141 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     4201 cccggcgaga agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc
     4261 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     4321 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     4381 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     4441 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     4501 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     4561 attttcttcg accagagcaa gaacggctac gccggctaca tcgatggcgg agccagccag
     4621 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     4681 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     4741 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     4801 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     4861 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     4921 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc cagcgcccag
     4981 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     5041 aagcacagcc tgctgtacga gtacttcacc gtgtacaacg agctgaccaa agtgaaatac
     5101 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa agccatcgtg
     5161 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     5221 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     5281 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     5341 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     5401 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     5461 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     5521 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     5581 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     5641 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     5701 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     5761 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     5821 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     5881 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     5941 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     6001 atcaaccggc tgtccgacta cgatgtggac gctatcgtgc ctcagagctt tctgaaggac
     6061 gactccatcg ataacaaagt gctgactcgg agcgacaaga accggggcaa gagcgacaac
     6121 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcgccagct gctgaatgcc
     6181 aagctgatta cccagaggaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     6241 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     6301 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa cgacaaactg
     6361 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     6421 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     6481 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     6541 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     6601 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     6661 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaaca
     6721 ggcgagatcg tgtgggataa gggccgggac tttgccaccg tgcggaaagt gctgtctatg
     6781 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     6841 atcctgccca agaggaacag cgacaagctg atcgccagaa agaaggactg ggaccctaag
     6901 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     6961 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     7021 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     7081 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     7141 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     7201 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     7261 gaggataatg agcagaaaca gctgtttgtg gaacagcaca aacactacct ggacgagatc
     7321 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaag
     7381 gtgctgagcg cctacaacaa gcacagagac aagcctatca gagagcaggc cgagaatatc
     7441 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     7501 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     7561 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacgcc
     7621 tatccctatg acgtgcccga ttatgccagc ctgggcagcg gctcccccaa gaaaaaacgc
     7681 aaggtggaag atcctaagaa aaagcggaaa gtggacggca ttggtagtgg gagcaacggc
     7741 agcagcggat ccaattgagg ccttaattaa tgtacaaggc ggccgcgtcg acaatcaacc
     7801 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     7861 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     7921 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     7981 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     8041 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     8101 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     8161 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     8221 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     8281 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     8341 ccctcagacg agtcggatct ccctttgggc cgcctccccg cacgcgtggc ctccgcgccg
     8401 ggttttggcg cctcccgcgg gcgcccccct cctcacggcg agcgctgcca cgtcagacga
     8461 agggcgcagc gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc
     8521 ataagactcg gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg
     8581 tgactctagg gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct
     8641 tctcggcgat tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga
     8701 cgcgccgggt gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt
     8761 tgtggatcgc tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc
     8821 gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt
     8881 ctgggtccgc gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg
     8941 gctgttcccg agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca
     9001 aggtgggggg catggtgggc ggcaagaacc caaggtcttg aggccttcgc taatgcggga
     9061 aagctcttat tcgggtgaga tgggctgggg caccatctgg ggaccctgac gtgaagtttg
     9121 tcactgactg gagaactcgg tttgtcgtct gttgcggggg cggcagttat ggcggtgccg
     9181 ttgggcagtg cacccgtacc tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg
     9241 ttctgttggc ttataatgca gggtggggcc acctgccggt aggtgtgcgg taggcttttc
     9301 tccgtcgcag gacgcagggt tcgggcctag ggtaggctct cctgaatcga caggcgccgg
     9361 acctctggtg aggggaggga taagtgaggc gtcagtttct ttggtcggtt ttatgtacct
     9421 atcttcttaa gtagctgaag ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt
     9481 tttgtgaagt tttttaggca ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc
     9541 agtgttagac tagtaaattg tccgctaaat tctggccgtt tttggctttt ttgttagacg
     9601 ctagcggatc cataacttcg tatagtatac attatacgaa gttatgccac catgtctagg
     9661 ctggacaaga gcaaagtcat aaacggagct ctggaattac tcaatggtgt cggtatcgaa
     9721 ggcctgacga caaggaaact cgctcaaaag ctgggagttg agcagcctac cctgtactgg
     9781 cacgtgaaga acaagcgggc cctgctcgat gccctgccaa tcgagatgct ggacaggcat
     9841 catacccact tctgccccct ggaaggcgag tcatggcaag actttctgcg gaacaacgcc
     9901 aagtcatacc gctgtgctct cctctcacat cgcgacgggg ctaaagtgca tctcggcacc
     9961 cgcccaacag agaaacagta cgaaaccctg gaaaatcagc tcgcgttcct gtgtcagcaa
    10021 ggcttctccc tggagaacgc actgtacgct ctgtccgccg tgggccactt tacactgggc
    10081 tgcgtattgg aggaacagga gcatcaagta gcaaaagagg aaagagagac acctaccacc
    10141 gattctatgc ccccacttct gagacaagca attgagctgt tcgaccggca gggagccgaa
    10201 cctgccttcc ttttcggcct ggaactaatc atatgtggcc tggagaaaca gctaaagtgc
    10261 gaaagcggcg ggccgaccga cgcccttgac gattttgact tagacatgct cccagccgat
    10321 gcccttgacg attttgacct tgacatgctc cccgggtaaa taacttcgta tagtatacat
    10381 tatacgaagt tatggatccg cggccgcaaa ttccgcccct ctccctcccc cccccctaac
    10441 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
    10501 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
    10561 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg
    10621 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
    10681 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
    10741 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
    10801 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta
    10861 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg
    10921 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca
    10981 cgataatacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
    11041 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
    11101 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
    11161 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
    11221 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
    11281 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
    11341 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggcgtcg acaatcaacc
    11401 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
    11461 gctatgtgga tacgctgctt taatgccttt gtatcatgcg ttaactaact aaacttgttt
    11521 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
    11581 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
    11641 tggaattgac tcaaatgatg tcaattagtc tatcagaagc tcatctggtc tcccttccgg
    11701 gggacaagac atccctgttt aatatttaaa cagcagtgtt cccaaactgg gttcttatat
    11761 cccttgctct ggtcaaccag gttgcagggt ttcctgtcct cacaggaacg aagtccctaa
    11821 agaaacagtg gcagccaggt ttagccccgg aattgactgg attccttttt tagggcccat
    11881 tggtatggct ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag
    11941 cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg
    12001 ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct
    12061 gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc
    12121 tgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat
    12181 aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc
    12241 attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca
    12301 cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc
    12361 gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat
    12421 ctttctaggg ttaatctagc tgcatcagga tcatatcgtc gggtcttttt tccggctcag
    12481 tcatcgccca agctggcgct atctgggcat cggggaggaa gaagcccgtg ccttttcccg
    12541 cgaggttgaa gcggcatgga aagagtttgc cgaggatgac tgctgctgca ttgacgttga
    12601 gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg gccatgcacg cctttaacgg
    12661 tgaactgttc gttcaggcca cctgggatac cagttcgtcg cggcttttcc ggacacagtt
    12721 ccggatggtc agcccgaagc gcatcagcaa cccgaacaat accggcgaca gccggaactg
    12781 ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg ggatattacg tcagcgagga
    12841 cgggtatcct ggctggatgc cgcagaaatg gacatggata ccccgtgagt tacccggcgg
    12901 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
    12961 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
    13021 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
    13081 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
    13141 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
    13201 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
    13261 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
    13321 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
    13381 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
    13441 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
    13501 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
    13561 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
    13621 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
    13681 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
    13741 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
    13801 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
    13861 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
    13921 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
    13981 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
    14041 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
    14101 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
    14161 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
    14221 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
    14281 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
    14341 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
    14401 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
    14461 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
    14521 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
    14581 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
    14641 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtca
//
