LOCUS       sequence-233395-       13424 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SP-dCas9-KRAB-MeCP2 with doxycycline-inducible expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13424)
  AUTHORS   Tan X, Worley J, Turunen M, Wong K, Fernandez EC, Paull E, Jones S, 
            Wang J, Noh H, Salvatori B, Chavez A, Califano A
  TITLE     Interrogation of genome-wide, experimentally dissected gene 
            regulatory networks reveals mechanisms underlying dynamic cellular 
            state control
  JOURNAL   bioRxiv 2021.06.28.449297
REFERENCE   2  (bases 1 to 13424)
  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: "bioRxiv 
            2021.06.28.449297"
COMMENT     Sequence Label: PB-TRE-dCas9-KRAB-MeCP2
FEATURES             Location/Qualifiers
     source          1..13424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(14..469)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(151..172)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     363..382
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     596..618
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     610..627
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     611..627
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   757..819
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1268..1646
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1276..1294
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1312..1330
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1348..1366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1384..1402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1420..1438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1456..1474
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1492..1510
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1555..1579
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1673..1682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1679..1705,1706..1708,1709..4192,4193..4198,4199..4264,
                     4265..4267,4268..5782)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5786..5812
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5792..5818
                     /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             5831..5851
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5858..5878
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5918..6103
                     /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             6137..6157
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6167..7024
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     primer_bind     complement(7079..7098)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7123..7171
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7402..7613
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             7626..7894
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             7928..8671
                     /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"
     CDS             8672..8725
                     /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             8747..9766
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             8747..9766
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(9823..9843)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    9877..9998
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9914..9933)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9968..9987
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10761..10795
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11286..11302)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11286..11302)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11299..11321)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11310..11326
                     /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(11334..11340,11341..11358,11359..11364))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11379..11400
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11517..11534)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11688..12276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11768..11787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12447..13238,13239..13307))
                     /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"
     primer_bind     13070..13089
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13308..13412)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
       61 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
      121 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
      181 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
      241 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
      301 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
      361 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
      421 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
      481 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
      541 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      601 tcacgacgtt gtaaaacgac ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc
      661 gtggaggacg ggcagactcg cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag
      721 atgctcgaca cgctgcagaa cacgcagcta gattaaccct agaaagataa tcatattgtg
      781 acgtacgtta aagataatca tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc
      841 gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac ttacatacta
      901 ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa aacaaaaact
      961 caaaatttct tctataaagt aacaaaactt ttatgaggga cagccccccc ccaaagcccc
     1021 cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg
     1081 ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca
     1141 cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc
     1201 agacacctgg ggggatacgg ggaaaaggcc tccacggcca ctagtctttc gtcttcaaga
     1261 attcctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
     1321 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
     1381 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
     1441 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
     1501 gtgatagaga acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg
     1561 tttagtgaac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt
     1621 tccgtaccac ttcctaccct cgtaaaggtc tagagctagc gaattcgaat ttgccaccat
     1681 ggacaagaag tactccattg ggctcgctat cggcacaaac agcgtcggct gggccgtcat
     1741 tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca
     1801 cagcataaag aagaacctca ttggcgccct cctgttcgac tccggggaga cggccgaagc
     1861 cacgcggctc aaaagaacag cacggcgcag atatacccgc agaaagaatc ggatctgcta
     1921 cctgcaggag atctttagta atgagatggc taaggtggat gactctttct tccataggct
     1981 ggaggagtcc tttttggtgg aggaggataa aaagcacgag cgccacccaa tctttggcaa
     2041 tatcgtggac gaggtggcgt accatgaaaa gtacccaacc atatatcatc tgaggaagaa
     2101 gcttgtagac agtactgata aggctgactt gcggttgatc tatctcgcgc tggcgcatat
     2161 gatcaaattt cggggacact tcctcatcga gggggacctg aacccagaca acagcgatgt
     2221 cgacaaactc tttatccaac tggttcagac ttacaatcag cttttcgaag agaacccgat
     2281 caacgcatcc ggagttgacg ccaaagcaat cctgagcgct aggctgtcca aatcccggcg
     2341 gctcgaaaac ctcatcgcac agctccctgg ggagaagaag aacggcctgt ttggtaatct
     2401 tatcgccctg tcactcgggc tgacccccaa ctttaaatct aacttcgacc tggccgaaga
     2461 tgccaagctt caactgagca aagacaccta cgatgatgat ctcgacaatc tgctggccca
     2521 gatcggcgac cagtacgcag accttttttt ggcggcaaag aacctgtcag acgccattct
     2581 gctgagtgat attctgcgag tgaacacgga gatcaccaaa gctccgctga gcgctagtat
     2641 gatcaagcgc tatgatgagc accaccaaga cttgactttg ctgaaggccc ttgtcagaca
     2701 gcaactgcct gagaagtaca aggaaatttt cttcgatcag tctaaaaatg gctacgccgg
     2761 atacattgac ggcggagcaa gccaggagga attttacaaa tttattaagc ccatcttgga
     2821 aaaaatggac ggcaccgagg agctgctggt aaagcttaac agagaagatc tgttgcgcaa
     2881 acagcgcact ttcgacaatg gaagcatccc ccaccagatt cacctgggcg aactgcacgc
     2941 tatcctcagg cggcaagagg atttctaccc ctttttgaaa gataacaggg aaaagattga
     3001 gaaaatcctc acatttcgga taccctacta tgtaggcccc ctcgcccggg gaaattccag
     3061 attcgcgtgg atgactcgca aatcagaaga gaccatcact ccctggaact tcgaggaagt
     3121 cgtggataag ggggcctctg cccagtcctt catcgaaagg atgactaact ttgataaaaa
     3181 tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg tacgagtact tcacagttta
     3241 taacgagctc accaaggtca aatacgtcac agaagggatg agaaagccag cattcctgtc
     3301 tggagagcag aagaaagcta tcgtggacct cctcttcaag acgaaccgga aagttaccgt
     3361 gaaacagctc aaagaagact atttcaaaaa gattgaatgt ttcgactctg ttgaaatcag
     3421 cggagtggag gatcgcttca acgcatccct gggaacgtat cacgatctcc tgaaaatcat
     3481 taaagacaag gacttcctgg acaatgagga gaacgaggac attcttgagg acattgtcct
     3541 cacccttacg ttgtttgaag atagggagat gattgaagaa cgcttgaaaa cttacgctca
     3601 tctcttcgac gacaaagtca tgaaacagct caagaggcgc cgatatacag gatgggggcg
     3661 gctgtcaaga aaactgatca atgggatccg agacaagcag agtggaaaga caatcctgga
     3721 ttttcttaag tccgatggat ttgccaaccg gaacttcatg cagttgatcc atgatgactc
     3781 tctcaccttt aaggaggaca tccagaaagc acaagtttct ggccaggggg acagtcttca
     3841 cgagcacatc gctaatcttg caggtagccc agctatcaaa aagggaatac tgcagaccgt
     3901 taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat aagcccgaga atatcgttat
     3961 cgagatggcc cgagagaacc aaactaccca gaagggacag aagaacagta gggaaaggat
     4021 gaagaggatt gaagagggta taaaagaact ggggtcccaa atccttaagg aacacccagt
     4081 tgaaaacacc cagcttcaga atgagaagct ctacctgtac tacctgcaga acggcaggga
     4141 catgtacgtg gatcaggaac tggacatcaa tcggctctcc gactacgacg tggctgctat
     4201 cgtgccccag tcttttctca aagatgattc tattgataat aaagtgttga caagatccga
     4261 taaagctaga gggaagagtg ataacgtccc ctcagaagaa gttgtcaaga aaatgaaaaa
     4321 ttattggcgg cagctgctga acgccaaact gatcacacaa cggaagttcg ataatctgac
     4381 taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc ggcttcatca aaaggcagct
     4441 tgttgagaca cgccagatca ccaagcacgt ggcccaaatt ctcgattcac gcatgaacac
     4501 caagtacgat gaaaatgaca aactgattcg agaggtgaaa gttattactc tgaagtctaa
     4561 gctggtctca gatttcagaa aggactttca gttttataag gtgagagaga tcaacaatta
     4621 ccaccatgcg catgatgcct acctgaatgc agtggtaggc actgcactta tcaaaaaata
     4681 tcccaagctt gaatctgaat ttgtttacgg agactataaa gtgtacgatg ttaggaaaat
     4741 gatcgcaaag tctgagcagg aaataggcaa ggccaccgct aagtacttct tttacagcaa
     4801 tattatgaat tttttcaaga ccgagattac actggccaat ggagagattc ggaagcgacc
     4861 acttatcgaa acaaacggag aaacaggaga aatcgtgtgg gacaagggta gggatttcgc
     4921 gacagtccgg aaggtcctgt ccatgccgca ggtgaacatc gttaaaaaga ccgaagtaca
     4981 gaccggaggc ttctccaagg aaagtatcct cccgaaaagg aacagcgaca agctgatcgc
     5041 acgcaaaaaa gattgggacc ccaagaaata cggcggattc gattctccta cagtcgctta
     5101 cagtgtactg gttgtggcca aagtggagaa agggaagtct aaaaaactca aaagcgtcaa
     5161 ggaactgctg ggcatcacaa tcatggagcg atcaagcttc gaaaaaaacc ccatcgactt
     5221 tctcgaggcg aaaggatata aagaggtcaa aaaagacctc atcattaagc ttcccaagta
     5281 ctctctcttt gagcttgaaa acggccggaa acgaatgctc gctagtgcgg gcgagctgca
     5341 gaaaggtaac gagctggcac tgccctctaa atacgttaat ttcttgtatc tggccagcca
     5401 ctatgaaaag ctcaaagggt ctcccgaaga taatgagcag aagcagctgt tcgtggaaca
     5461 acacaaacac taccttgatg agatcatcga gcaaataagc gaattctcca aaagagtgat
     5521 cctcgccgac gctaacctcg ataaggtgct ttctgcttac aataagcaca gggataagcc
     5581 catcagggag caggcagaaa acattatcca cttgtttact ctgaccaact tgggcgcgcc
     5641 tgcagccttc aagtacttcg acaccaccat agacagaaag cggtacacct ctacaaagga
     5701 ggtcctggac gccacactga ttcatcagtc aattacgggg ctctatgaaa caagaatcga
     5761 cctctctcag ctcggtggag acgcctatcc ctatgacgtg cccgattatg ccagcctggg
     5821 cagcggctcc cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc ggaaagtgga
     5881 cggcattggt agtgggagca acggcagcag cggatcccgg acactggtga ccttcaagga
     5941 tgtatttgtg gacttcacca gggaggagtg gaagctgctg gacactgctc agcagatcgt
     6001 gtacagaaat gtgatgctgg agaactataa gaacctggtt tccttgggtt atcagcttac
     6061 taagccagat gtgatcctcc ggttggagaa gggagaagag ccctcgggag gtggttcggg
     6121 tggctctgga tcaagcccaa agaagaaacg gaaggtggaa gcctcagtgc aggtgaaaag
     6181 ggtgctggaa aaatcccccg gcaaactcct cgtgaagatg cccttccagg cttcccctgg
     6241 cggaaaaggt gaagggggtg gcgcaaccac atctgcccag gtcatggtca tcaagcgacc
     6301 tggaaggaaa agaaaggccg aggctgaccc tcaggccatt ccaaagaaac ggggacgcaa
     6361 gccagggtcc gtggtcgcag ctgcagcagc tgaggctaag aaaaaggcag tgaaggaaag
     6421 ctccatccgc agtgtgcagg agactgtcct gcccatcaag aagaggaaga ctagggagac
     6481 cgtgtccatc gaggtcaaag aagtggtcaa gcccctgctc gtgtccaccc tgggcgaaaa
     6541 atctggaaag gggctcaaaa catgcaagtc acctggacgg aaaagcaagg agtctagtcc
     6601 aaaggggcgc tcaagctccg cttctagtcc ccctaaaaag gaacaccatc accatcacca
     6661 tcacgccgag tctcctaagg ctcctatgcc actgctccca ccacctccac cacctgagcc
     6721 acagtcaagc gaagacccca tcagcccacc cgagcctcag gatctgtcct ctagtatttg
     6781 caaagaggaa aagatgccca gagcaggcag cctggagagt gatggctgtc caaaagaacc
     6841 cgccaagacc cagcctatgg tggcagccgc tgcaactacc accacaacca caactaccac
     6901 agtggccgaa aaatacaagc atcgcggcga gggcgaacga aaggacattg tgtcaagctc
     6961 catgcccaga cctaaccggg aggaaccagt cgatagtagg acacccgtga ctgagagagt
     7021 ctcatgaaag ggttcgatcc ctaccggtta gtaatgagtt taaacggggg aggctaactg
     7081 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     7141 ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     7201 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     7261 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     7321 gcaggccctg ccatagcaga tctgcgaaat cggatccagc taaatctaag tggatctgcg
     7381 atcgctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     7441 ggggaggggt cggcaattga acgggtgcct agagaaggtg gcgcggggta aactgggaaa
     7501 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     7561 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca cagctgaagc
     7621 ttcgaggggc tcgcatctct ccttcacgcg cccgccgccc tacctgaggc cgccatccac
     7681 gccggttgag tcgcgttctg ccgcctcccg cctgtggtgc ctcctgaact gcgtccgccg
     7741 tctaggtaag tttaaagctc aggtcgagac cgggcctttg tccggcgctc ccttggagcc
     7801 tacctagact cagccggctc tccacgcttt gcctgaccct gcttgctcaa ctctacgtct
     7861 ttgtttcgtt ttctgttctg cgccgttaca gatctaagct gtgaccggcg cctactctag
     7921 agccgccatg tctagactgg acaagagcaa agtcataaac tctgctctgg aattactcaa
     7981 tggagtcggt atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca
     8041 gcctaccctg tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga
     8101 gatgctggac aggcatcata cccactcctg ccccctggaa ggcgagtcat ggcaagactt
     8161 tctgcggaac aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa
     8221 agtgcatctc ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc
     8281 gttcctgtgt cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg
     8341 ccactttaca ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag
     8401 agagacacct accaccgatt ctatgccccc acttctgaaa caagcaattg agctgttcga
     8461 ccggcaggga gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga
     8521 gaaacagcta aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga
     8581 catgctccca gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct
     8641 tgacgatttt gaccttgaca tgctccccgg ggagggcaga ggaagtcttc taacatgcgg
     8701 tgacgtggag gagaatcccg gccctgaatt catggataga tccggaaagc ctgaactcac
     8761 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     8821 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     8881 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     8941 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     9001 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     9061 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     9121 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     9181 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     9241 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     9301 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     9361 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     9421 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     9481 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     9541 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     9601 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     9661 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     9721 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataagcca aggaacctct
     9781 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     9841 atgtggatac gctgctttaa tgcctttgta tcagttaact tgtttattgc agcttataat
     9901 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     9961 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggaa ttgactcaaa
    10021 tgatgtcaat tagtctatca gaagctcatc tggtctccct tccgggggac aagacatccc
    10081 tgtttaatat ttaaacagca gtgttcccaa actgggttct tatatccctt gctctggtca
    10141 accaggttgc agggtttcct gtcctcacag gaacgaagtc cctaaagaaa cagtggcagc
    10201 caggtttagc cccggaattg actggattcc ttttttaggg cccattggta tggctttttc
    10261 cccgtatccc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc
    10321 gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc
    10381 ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg
    10441 agggacgtaa ttacatccct gggggctttg ggggggggct gtccctgata tctataacaa
    10501 gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa tataattatc
    10561 gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg
    10621 gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct
    10681 ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag
    10741 agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaat
    10801 ctagctgcat caggatcata tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg
    10861 gcgctatctg ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc
    10921 atggaaagag tttgccgagg atgactgctg ctgcattgac gttgagcgaa aacgcacgtt
    10981 taccatgatg attcgggaag gtgtggccat gcacgccttt aacggtgaac tgttcgttca
    11041 ggccacctgg gataccagtt cgtcgcggct tttccggaca cagttccgga tggtcagccc
    11101 gaagcgcatc agcaacccga acaataccgg cgacagccgg aactgccgtg ccggtgtgca
    11161 gattaatgac agcggtgcgg cgctgggata ttacgtcagc gaggacgggt atcctggctg
    11221 gatgccgcag aaatggacat ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa
    11281 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
    11341 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
    11401 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
    11461 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
    11521 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
    11581 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
    11641 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
    11701 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
    11761 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
    11821 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
    11881 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
    11941 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
    12001 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
    12061 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
    12121 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
    12181 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
    12241 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
    12301 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
    12361 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
    12421 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
    12481 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
    12541 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
    12601 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
    12661 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
    12721 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
    12781 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
    12841 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
    12901 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
    12961 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
    13021 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
    13081 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
    13141 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
    13201 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
    13261 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
    13321 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
    13381 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccg
//
