LOCUS       sequence-331121-       14511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible CRISPRa expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14511)
  AUTHORS   Sun D, Evans L, Perrone F, Sokleva V, Lim K, Rezakhani S, Lutolf M, 
            Zilbauer M, Rawlins EL
  TITLE     A functional genetic toolbox for human tissue-derived organoids.
  JOURNAL   eLife. 2021 Oct 6;10. pii: 67886. doi: 10.7554/eLife.67886.
  PUBMED    34612202
REFERENCE   2  (bases 1 to 14511)
  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: "2021-10-6"; volume: "10. pii: 67886. doi"; pages: " 
            10.7554/eLife.67886"
COMMENT     Sequence Label: pLenti-tetON-dxCas9(3.7)-VPR-EF1a-TagRFP-2A-tet3G
FEATURES             Location/Qualifiers
     source          1..14511
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2246..2264
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2282..2300
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2318..2336
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2354..2372
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2390..2408
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2426..2444
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2462..2480
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2525..2549
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2672..2681
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2678..6781
                     /codon_start=1
                     /product="catalytically dead Streptococcus pyogenes Cas9 
                     mutant with broad PAM compatibility (Hu et al., 2018)"
                     /label=dxCas9(3.7)
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6794..6814
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    6851..6875
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6905..7054
                     /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             7079..7099
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7529..7885
                     /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             7904..8473
                     /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"
     primer_bind     complement(8514..8533)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    8579..8634
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(8633..8652)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        8838..9049
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             9062..9330
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      9365..9374
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             9371..10081
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             10088..10141
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             10142..10888
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    10901..11489
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10954..10974)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11372..11383)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11564..11797
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(11916..11934)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        12002..12106
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12107..12175,12176..12967)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(12325..12344)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13138..13726
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13627..13646
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13880..13897
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    14137..14158
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(14173..14178,14179..14196,14197..14203)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14211..14227
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     14216..14238
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     14235..14251
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     14235..14251
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     14301..14320
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagaccg
     2221 gtggcctcca cggccactag attactccct atcagtgata gagaacgtat gaagagttta
     2281 ctccctatca gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt
     2341 ataaggagtt tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt
     2401 gatagagaac gtatctacag tttactccct atcagtgata gagaacgtat atccagttta
     2461 ctccctatca gtgatagaga acgtataagc tttaggcgtg tacggtgggc gcctataaaa
     2521 gcagagctcg tttagtgaac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt
     2581 ataccaactt tccgtaccac ttcctaccct cgtaaagaag ttcctattcc gaagttccta
     2641 ttcttcaaaa ggtataggaa cttcgctcga ggccaccatg gacaagaagt actccattgg
     2701 gctcgctatc ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc
     2761 gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat
     2821 tggcgccctc ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc
     2881 acggcgcaga tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa
     2941 tgagatggct aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga
     3001 ggaggataaa aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta
     3061 ccatgaaaag tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa
     3121 ggctgacttg cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt
     3181 cctcatcgag ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact
     3241 ggttcagact tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc
     3301 caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca
     3361 gctccctggg gagaagaaga acggcctgtt tggtaatctt atcgccctgt ccctcgggct
     3421 gacccccaac tttaaatcta acttcgacct ggccgaagat accaagcttc aactgagcaa
     3481 agacacctac gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga
     3541 cctttttttg gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt
     3601 gaacacggag atcaccaaag ctccgctgag cgctagtatg atcaagctct atgatgagca
     3661 ccaccaagac ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa
     3721 ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag
     3781 ccaggaggaa ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga
     3841 gctgctggta aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg
     3901 aatcatcccc caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga
     3961 tttctacccc tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat
     4021 accctactat gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa
     4081 atcagaagag accatcactc cctggaactt cgagaaagtc gtggataagg gggcctctgc
     4141 ccagtccttc atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct
     4201 tcctaaacac tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa
     4261 atacgtcaca gaagggatga gaaagccagc attcctgtct ggagatcaga agaaagctat
     4321 tgtggacctc ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta
     4381 tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa
     4441 cgcatccctg ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga
     4501 caatgaggag aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga
     4561 tagggagatg attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat
     4621 gaagcagctc aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa
     4681 tgggatccga gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt
     4741 tgccaaccgg aacttcattc agttgatcca tgatgactct ctcaccttta aggaggacat
     4801 ccagaaagca caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc
     4861 aggtagccca gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt
     4921 caaagtaatg ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca
     4981 aaccacccag aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat
     5041 aaaagaactg gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa
     5101 tgagaagctc tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact
     5161 ggacatcaat cggctctccg actacgacgt ggacgctatc gtgccccagt cttttctcaa
     5221 agatgattct attgataata aagtgttgac aagatccgat aaaaacagag ggaagagtga
     5281 taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa
     5341 cgccaaactg atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct
     5401 gtctgagttg gataaagccg gtttcatcaa aaggcagctt gttgagacac gccagatcac
     5461 caagcacgtg gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa
     5521 actgattcga gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa
     5581 ggactttcag ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta
     5641 cctgaatgca gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt
     5701 tgtttacgga gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga
     5761 aataggcaag gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac
     5821 cgagattaca ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga
     5881 aacaggagaa atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc
     5941 catgccgcag gtgaacatcg ttaaaaagac cgaagtacag accggaggtt tctccaagga
     6001 aagtatcctc ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc
     6061 caagaaatac ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa
     6121 agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat
     6181 catggagcga tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa
     6241 agaggtcaaa aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa
     6301 cggccggaaa cgaatgctcg ctagtgcggg cgtgctgcag aaaggtaacg agctggcact
     6361 gccctctaaa tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc
     6421 tcccgaagat aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga
     6481 gatcatcgag caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga
     6541 taaggtgctt tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa
     6601 cattatccac ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga
     6661 cactaccata gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat
     6721 tcatcagtca attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga
     6781 cagcagggct gaccccaaga agaagaggaa ggtgtcgcca gggatccgtc gacttgacgc
     6841 gttgatatca acaagtttgt acaaaaaagc aggctacaaa gaggccagcg gttccggacg
     6901 ggctgacgca ttggacgatt ttgatctgga tatgctggga agtgacgccc tcgatgattt
     6961 tgaccttgac atgcttggtt cggatgccct tgatgacttt gacctcgaca tgctcggcag
     7021 tgacgccctt gatgatttcg acctggacat gctgattaac tctagaagtt ccggatctcc
     7081 gaaaaagaaa cgcaaagttg gtagccagta cctgcccgac accgacgacc ggcaccggat
     7141 cgaggaaaag cggaagcgga cctacgagac attcaagagc atcatgaaga agtccccctt
     7201 cagcggcccc accgacccta gacctccacc tagaagaatc gccgtgccca gcagatccag
     7261 cgccagcgtg ccaaaacctg ccccccagcc ttaccccttc accagcagcc tgagcaccat
     7321 caactacgac gagttcccta ccatggtgtt ccccagcggc cagatctctc aggcctctgc
     7381 tctggctcca gcccctcctc aggtgctgcc tcaggctcct gctcctgcac cagctccagc
     7441 catggtgtct gcactggctc aggcaccagc acccgtgcct gtgctggctc ctggacctcc
     7501 acaggctgtg gctccaccag cccctaaacc tacacaggcc ggcgagggca cactgtctga
     7561 agctctgctg cagctgcagt tcgacgacga ggatctggga gccctgctgg gaaacagcac
     7621 cgatcctgcc gtgttcaccg acctggccag cgtggacaac agcgagttcc agcagctgct
     7681 gaaccagggc atccctgtgg cccctcacac caccgagccc atgctgatgg aataccccga
     7741 ggccatcacc cggctcgtga caggcgctca gaggcctcct gatccagctc ctgcccctct
     7801 gggagcacca ggcctgccta atggactgct gtctggcgac gaggacttca gctctatcgc
     7861 cgatatggat ttctcagcct tgctgggctc tggcagcggc agccgggatt ccagggaagg
     7921 gatgtttttg ccgaagcctg aggccggctc cgctattagt gacgtgtttg agggccgcga
     7981 ggtgtgccag ccaaaacgaa tccggccatt tcatcctcca ggaagtccat gggccaaccg
     8041 cccactcccc gccagcctcg caccaacacc aaccggtcca gtacatgagc cagtcgggtc
     8101 actgaccccg gcaccagtcc ctcagccact ggatccagcg cccgcagtga ctcccgaggc
     8161 cagtcacctg ttggaggatc ccgatgaaga gacgagccag gctgtcaaag cccttcggga
     8221 gatggccgat actgtgattc cccagaagga agaggctgca atctgtggcc aaatggacct
     8281 ttcccatccg cccccaaggg gccatctgga tgagctgaca accacacttg agtccatgac
     8341 cgaggatctg aacctggact cacccctgac cccggaattg aacgagattc tggatacctt
     8401 cctgaacgac gagtgcctct tgcatgccat gcatatcagc acaggactgt ccatcttcga
     8461 cacatctctg ttttgaggat ccaccggatc tagataactg agcggccgcc taatcttttt
     8521 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa
     8581 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga
     8641 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg
     8701 gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag
     8761 tatatgaaac agccccctgc tgtccattcc ttacatatga ctagtaagga tctgcgatcg
     8821 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     8881 aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     8941 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     9001 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg
     9061 aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg
     9121 gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta
     9181 ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc
     9241 tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt
     9301 ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta cacctgcagc
     9361 caacgccacc atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct
     9421 gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa
     9481 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     9541 cgccttcgac atcctggcta ccagcttcat gtacggcagc agaaccttca tcaaccacac
     9601 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt
     9661 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     9721 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat
     9781 gcagaagaaa acactcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg
     9841 cctggaaggc agaagcgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa
     9901 cttcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta
     9961 ctatgtggac cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca
    10021 gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa
    10081 tgctagcgag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
    10141 aatgtctaga ctggacaaga gcaaagtcat aaactctgct ctggaattac tcaatggagt
    10201 cggtatcgaa ggcctgacga caaggaaact cgctcaaaag ctgggagttg agcagcctac
    10261 cctgtactgg cacgtgaaga acaagcgggc cctgctcgat gccctgccaa tcgagatgct
    10321 ggacaggcat catacccact cctgccccct ggaaggcgag tcatggcaag actttctgcg
    10381 gaacaacgcc aagtcatacc gctgtgctct cctctcacat cgcgacgggg ctaaagtgca
    10441 tctcggcacc cgcccaacag agaaacagta cgaaaccctg gaaaatcagc tcgcgttcct
    10501 gtgtcagcaa ggcttctccc tggagaacgc actgtacgct ctgtccgccg tgggccactt
    10561 tacactgggc tgcgtattgg aggaacagga gcatcaagta gcaaaagagg aaagagagac
    10621 acctaccacc gattctatgc ccccacttct gaaacaagca attgagctgt tcgaccggca
    10681 gggagccgaa cctgccttcc ttttcggcct ggaactaatc atatgtggcc tggagaaaca
    10741 gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac gattttgact tagacatgct
    10801 cccagccgat gcccttgacg actttgacct tgatatgctg cctgctgacg ctcttgacga
    10861 ttttgacctt gacatgctcc ccgggtaagt cgacgatcct aatcaacctc tggattacaa
    10921 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
    10981 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
    11041 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
    11101 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
    11161 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
    11221 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
    11281 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
    11341 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
    11401 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
    11461 tcggatctcc ctttgggccg cctccccgcc tgagatcctt taagaccaat gacttacaag
    11521 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
    11581 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
    11641 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
    11701 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
    11761 ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt
    11821 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggcc cgggttaatt
    11881 aaggaaaggg ctagatcatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
    11941 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
    12001 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
    12061 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
    12121 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
    12181 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
    12241 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
    12301 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
    12361 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
    12421 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
    12481 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
    12541 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
    12601 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
    12661 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
    12721 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
    12781 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
    12841 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
    12901 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
    12961 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
    13021 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
    13081 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
    13141 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
    13201 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
    13261 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
    13321 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
    13381 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
    13441 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
    13501 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
    13561 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
    13621 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
    13681 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
    13741 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
    13801 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
    13861 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
    13921 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agcaagctca tggctgacta
    13981 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
    14041 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tccccgtggc acgacaggtt
    14101 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
    14161 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
    14221 ataacaattt cacacaggaa acagctatga catgattacg aatttcacaa ataaagcatt
    14281 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
    14341 gatcaactgg ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat
    14401 taccactgcc aattacctgt ggtttcattt actctaaacc tgtgattcct ctgaattatt
    14461 ttcattttaa agaaattgta tttgttaaat atgtactaca aacttagtag t
//
