LOCUS       sequence-331141-       13647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible CRISPRi expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13647)
  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 13647)
  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-KRAB-dCas9-DHFR-EF1a-TagRFP-2A-tet3G
FEATURES             Location/Qualifiers
     source          1..13647
                     /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             2708..2893
                     /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             join(2915..2941,2942..2944,2945..5431,5432..5434,
                     5435..7018)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2915..7018
                     /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
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7022..7048
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7028..7054
                     /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             7067..7087
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7094..7114
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7136..7612
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     primer_bind     complement(7650..7669)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    7715..7770
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7769..7788)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        7974..8185
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8198..8466
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      8501..8510
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8507..9217
                     /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             9224..9277
                     /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             9278..10024
                     /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    10037..10625
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10090..10110)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10508..10519)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10700..10933
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(11052..11070)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        11138..11242
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11243..11311,11312..12103)
                     /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(11461..11480)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12274..12862
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12763..12782
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13016..13033
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13273..13294
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13309..13314,13315..13332,13333..13339)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13347..13363
                     /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     13352..13374
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13371..13387
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13371..13387
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13437..13456
                     /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 gatgctaagt cactaactgc
     2701 ctggtcccgg acactggtga ccttcaagga tgtatttgtg gacttcacca gggaggagtg
     2761 gaagctgctg gacactgctc agcagatcgt gtacagaaat gtgatgctgg agaactataa
     2821 gaacctggtt tccttgggtt atcagcttac taagccagat gtgatcctcc ggttggagaa
     2881 gggagaagag cccggcggtt ccggcggagg gtcgatggac aagaagtaca gcatcggcct
     2941 ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
     3001 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
     3061 cgccctgctg ttcgacagcg gagaaacagc cgaggccacc cggctgaaga gaaccgccag
     3121 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga
     3181 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga
     3241 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca
     3301 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
     3361 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct
     3421 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt
     3481 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa
     3541 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct
     3601 gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac
     3661 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
     3721 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
     3781 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa
     3841 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca
     3901 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga
     3961 gattttcttc gaccagagca agaacggcta cgccggctac atcgatggcg gagccagcca
     4021 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
     4081 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag
     4141 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt
     4201 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc
     4261 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag
     4321 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg ccagcgccca
     4381 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc
     4441 caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca aagtgaaata
     4501 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aagccatcgt
     4561 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt
     4621 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc
     4681 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa
     4741 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag
     4801 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa
     4861 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg
     4921 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc
     4981 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca
     5041 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg
     5101 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
     5161 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac
     5221 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa
     5281 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga
     5341 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga
     5401 catcaaccgg ctgtccgact acgatgtgga cgctatcgtg cctcagagct ttctgaagga
     5461 cgactccatc gataacaaag tgctgactcg gagcgacaag aaccggggca agagcgacaa
     5521 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgccagc tgctgaatgc
     5581 caagctgatt acccagagga agttcgacaa tctgaccaag gccgagagag gcggcctgag
     5641 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa
     5701 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga acgacaaact
     5761 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga
     5821 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
     5881 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
     5941 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat
     6001 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga
     6061 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac
     6121 aggcgagatc gtgtgggata agggccggga ctttgccacc gtgcggaaag tgctgtctat
     6181 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
     6241 tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact gggaccctaa
     6301 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt
     6361 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat
     6421 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga
     6481 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg
     6541 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc
     6601 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc
     6661 cgaggataat gagcagaaac agctgtttgt ggaacagcac aaacactacc tggacgagat
     6721 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa
     6781 ggtgctgagc gcctacaaca agcacagaga caagcctatc agagagcagg ccgagaatat
     6841 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac
     6901 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
     6961 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacgc
     7021 ctatccctat gacgtgcccg attatgccag cctgggcagc ggctccccca agaaaaaacg
     7081 caaggtggaa gatcctaaga aaaagcggaa agtgggatcc gggggagcag gaggcatcag
     7141 tctgattgcg gcgttagcgg tagatcacgt tatcggcatg gaaaccgtca tgccgtggaa
     7201 cctgcctgcc gatctcgcct ggtttaaacg caacacctta aataaacccg tgattatggg
     7261 ccgccatacc tgggaatcaa tcggtcgtcc gttgccagga cgcaaaaata ttatcctcag
     7321 cagtcaaccg agtacggacg atcgcgtaac gtgggtgaag tcggtggatg aagccatcgc
     7381 ggcgtgtggt gacgtaccag aaatcatggt tattggcggc ggtcgcgttt atgaacagtt
     7441 cttgccaaaa gcgcaaaaac tgtatctgac gcatatcgac gcagaagtgg aaggcgacac
     7501 ccatttcccg gattacgagc cggatgactg ggaatcggta ttcagcgaat tccacgatgc
     7561 tgatgcgcag aactctcaca gctattgctt tgagattctg gagcggcgat gaggatccac
     7621 cggatctaga taactgagcg gccgcctaat ctttttccct ctgccaaaaa ttatggggac
     7681 atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca
     7741 atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat
     7801 ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca tatgctggct
     7861 gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc
     7921 cattccttac atatgactag taaggatctg cgatcgctcc ggtgcccgtc agtgggcaga
     7981 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaacgggtgc
     8041 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     8101 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     8161 caacgggttt gccgccagaa cacagctgaa gcttcgaggg gctcgcatct ctccttcacg
     8221 cgcccgccgc cctacctgag gccgccatcc acgccggttg agtcgcgttc tgccgcctcc
     8281 cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc tcaggtcgag
     8341 accgggcctt tgtccggcgc tcccttggag cctacctaga ctcagccggc tctccacgct
     8401 ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg ttttctgttc tgcgccgtta
     8461 cagatccaag ctgtgaccgg cgcctacacc tgcagccaac gccaccatgg tgtctaaggg
     8521 cgaagagctg attaaggaga acatgcacat gaagctgtac atggagggca ccgtgaacaa
     8581 ccaccacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat
     8641 gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag
     8701 cttcatgtac ggcagcagaa ccttcatcaa ccacacccag ggcatccccg acttctttaa
     8761 gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt
     8821 gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat
     8881 cagaggggtg aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga
     8941 ggccaacacc gagatgctgt accccgctga cggcggcctg gaaggcagaa gcgacatggc
     9001 cctgaagctc gtgggcgggg gccacctgat ctgcaacttc aagaccacat acagatccaa
     9061 gaaacccgct aagaacctca agatgcccgg cgtctactat gtggaccaca gactggaaag
     9121 aatcaaggag gccgacaaag agacctacgt cgagcagcac gaggtggctg tggccagata
     9181 ctgcgacctc cctagcaaac tggggcacaa acttaatgct agcgagggca gaggaagtct
     9241 gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg tctagactgg acaagagcaa
     9301 agtcataaac tctgctctgg aattactcaa tggagtcggt atcgaaggcc tgacgacaag
     9361 gaaactcgct caaaagctgg gagttgagca gcctaccctg tactggcacg tgaagaacaa
     9421 gcgggccctg ctcgatgccc tgccaatcga gatgctggac aggcatcata cccactcctg
     9481 ccccctggaa ggcgagtcat ggcaagactt tctgcggaac aacgccaagt cataccgctg
     9541 tgctctcctc tcacatcgcg acggggctaa agtgcatctc ggcacccgcc caacagagaa
     9601 acagtacgaa accctggaaa atcagctcgc gttcctgtgt cagcaaggct tctccctgga
     9661 gaacgcactg tacgctctgt ccgccgtggg ccactttaca ctgggctgcg tattggagga
     9721 acaggagcat caagtagcaa aagaggaaag agagacacct accaccgatt ctatgccccc
     9781 acttctgaaa caagcaattg agctgttcga ccggcaggga gccgaacctg ccttcctttt
     9841 cggcctggaa ctaatcatat gtggcctgga gaaacagcta aagtgcgaaa gcggcgggcc
     9901 gaccgacgcc cttgacgatt ttgacttaga catgctccca gccgatgccc ttgacgactt
     9961 tgaccttgat atgctgcctg ctgacgctct tgacgatttt gaccttgaca tgctccccgg
    10021 gtaagtcgac gatcctaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
    10081 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
    10141 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
    10201 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
    10261 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
    10321 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
    10381 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
    10441 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
    10501 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
    10561 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
    10621 cccgcctgag atcctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
    10681 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
    10741 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
    10801 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
    10861 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
    10921 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag
    10981 aaatgaatat cagagagtga gaggcccggg ttaattaagg aaagggctag atcattcttg
    11041 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
    11101 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
    11161 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
    11221 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
    11281 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
    11341 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
    11401 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
    11461 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
    11521 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
    11581 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
    11641 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
    11701 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
    11761 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
    11821 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
    11881 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
    11941 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
    12001 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
    12061 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
    12121 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
    12181 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
    12241 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
    12301 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
    12361 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
    12421 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
    12481 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
    12541 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
    12601 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
    12661 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
    12721 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
    12781 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
    12841 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
    12901 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
    12961 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
    13021 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
    13081 gccgattcat taatgcagca agctcatggc tgactaattt tttttattta tgcagaggcc
    13141 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
    13201 ggcttttgca aaaagctccc cgtggcacga caggtttccc gactggaaag cgggcagtga
    13261 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
    13321 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
    13381 ctatgacatg attacgaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
    13441 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc aactggataa ctcaagctaa
    13501 ccaaaatcat cccaaacttc ccaccccata ccctattacc actgccaatt acctgtggtt
    13561 tcatttactc taaacctgtg attcctctga attattttca ttttaaagaa attgtatttg
    13621 ttaaatatgt actacaaact tagtagt
//
