LOCUS       sequence-156108-       13808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SP-dCas9-VPR with doxycycline-inducible expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13808)
  AUTHORS   Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin 
            S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, 
            Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, 
            Church GM
  TITLE     Highly efficient Cas9-mediated transcriptional programming.
  JOURNAL   Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312.
  PUBMED    25730490
REFERENCE   2  (bases 1 to 13808)
  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: "Nat 
            Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312."
COMMENT     Sequence Label: PB-TRE-dCas9-VPR
FEATURES             Location/Qualifiers
     source          1..13808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1386..1764
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1394..1412
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1430..1448
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1466..1484
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1502..1520
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1538..1556
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1574..1592
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1610..1628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1673..1697
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1791..1800
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1797..1823,1824..1826,1827..4310,4311..4316,4317..4382,
                     4383..4385,4386..5900)
                     /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             5913..5933
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5958..6107
                     /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             6132..6152
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6582..6938
                     /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             6957..7526
                     /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(7581..7600)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7625..7673
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7904..8115
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8128..8396
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             8430..9173
                     /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             9174..9227
                     /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             9249..10268
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             9249..10268
                     /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(10325..10345)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    10379..10500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10416..10435)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10470..10489
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   11262..11296
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11787..11803)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11787..11803)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11800..11822)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11811..11827
                     /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(11835..11841,11842..11859,11860..11865))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11880..11901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12018..12035)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12189..12777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12269..12288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12948..13739,13740..13808))
                     /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     13571..13590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca
     1081 aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca
     1141 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct
     1201 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg
     1261 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
     1321 acacctgggg ggatacgggg aaaaggcctc cacggccact agtctttcgt cttcaagaat
     1381 tcctcgagtt tactccctat cagtgataga gaacgtatga agagtttact ccctatcagt
     1441 gatagagaac gtatgcagac tttactccct atcagtgata gagaacgtat aaggagttta
     1501 ctccctatca gtgatagaga acgtatgacc agtttactcc ctatcagtga tagagaacgt
     1561 atctacagtt tactccctat cagtgataga gaacgtatat ccagtttact ccctatcagt
     1621 gatagagaac gtataagctt taggcgtgta cggtgggcgc ctataaaagc agagctcgtt
     1681 tagtgaaccg tcagatcgcc tggagcaatt ccacaacact tttgtcttat accaactttc
     1741 cgtaccactt cctaccctcg taaaggtcta gagctagcga attcgaattt gccaccatgg
     1801 acaagaagta ctccattggg ctcgctatcg gcacaaacag cgtcggctgg gccgtcatta
     1861 cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc gatcgccaca
     1921 gcataaagaa gaacctcatt ggcgccctcc tgttcgactc cggggagacg gccgaagcca
     1981 cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg atctgctacc
     2041 tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc cataggctgg
     2101 aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc tttggcaata
     2161 tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg aggaagaagc
     2221 ttgtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg gcgcatatga
     2281 tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac agcgatgtcg
     2341 acaaactctt tatccaactg gttcagactt acaatcagct tttcgaagag aacccgatca
     2401 acgcatccgg agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa tcccggcggc
     2461 tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt ggtaatctta
     2521 tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg gccgaagatg
     2581 ccaagcttca actgagcaaa gacacctacg atgatgatct cgacaatctg ctggcccaga
     2641 tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac gccattctgc
     2701 tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc gctagtatga
     2761 tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt gtcagacagc
     2821 aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc tacgccggat
     2881 acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc atcttggaaa
     2941 aaatggacgg caccgaggag ctgctggtaa agcttaacag agaagatctg ttgcgcaaac
     3001 agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa ctgcacgcta
     3061 tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa aagattgaga
     3121 aaatcctcac atttcggata ccctactatg taggccccct cgcccgggga aattccagat
     3181 tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc ctggaacttc gaggaagtcg
     3241 tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt gataaaaatc
     3301 tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc acagtttata
     3361 acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca ttcctgtctg
     3421 gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa gttaccgtga
     3481 aacagctcaa agaagactat ttcaaaaaga ttgaatgttt cgactctgtt gaaatcagcg
     3541 gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg aaaatcatta
     3601 aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac attgtcctca
     3661 cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact tacgctcatc
     3721 tcttcgacga caaagtcatg aaacagctca agaggcgccg atatacagga tgggggcggc
     3781 tgtcaagaaa actgatcaat gggatccgag acaagcagag tggaaagaca atcctggatt
     3841 ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat gatgactctc
     3901 tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac agtcttcacg
     3961 agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg cagaccgtta
     4021 aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat atcgttatcg
     4081 agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg gaaaggatga
     4141 agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa cacccagttg
     4201 aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac ggcagggaca
     4261 tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg gctgctatcg
     4321 tgccccagtc ttttctcaaa gatgattcta ttgataataa agtgttgaca agatccgata
     4381 aagctagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa atgaaaaatt
     4441 attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat aatctgacta
     4501 aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa aggcagcttg
     4561 ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc atgaacacca
     4621 agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg aagtctaagc
     4681 tggtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc aacaattacc
     4741 accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc aaaaaatatc
     4801 ccaagcttga atctgaattt gtttacggag actataaagt gtacgatgtt aggaaaatga
     4861 tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt tacagcaata
     4921 ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg aagcgaccac
     4981 ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg gatttcgcga
     5041 cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc gaagtacaga
     5101 ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag ctgatcgcac
     5161 gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca gtcgcttaca
     5221 gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa agcgtcaagg
     5281 aactgctggg catcacaatc atggagcgat caagcttcga aaaaaacccc atcgactttc
     5341 tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctt cccaagtact
     5401 ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc gagctgcaga
     5461 aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg gccagccact
     5521 atgaaaagct caaagggtct cccgaagata atgagcagaa gcagctgttc gtggaacaac
     5581 acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa agagtgatcc
     5641 tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg gataagccca
     5701 tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg ggcgcgcctg
     5761 cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct acaaaggagg
     5821 tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca agaatcgacc
     5881 tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag gtggaggcca
     5941 gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg ggaagtgacg
     6001 ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac tttgacctcg
     6061 acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt aactctagaa
     6121 gttccggatc tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc gacaccgacg
     6181 accggcaccg gatcgaggaa aagcggaagc ggacctacga gacattcaag agcatcatga
     6241 agaagtcccc cttcagcggc cccaccgacc ctagacctcc acctagaaga atcgccgtgc
     6301 ccagcagatc cagcgccagc gtgccaaaac ctgcccccca gccttacccc ttcaccagca
     6361 gcctgagcac catcaactac gacgagttcc ctaccatggt gttccccagc ggccagatct
     6421 ctcaggcctc tgctctggct ccagcccctc ctcaggtgct gcctcaggct cctgctcctg
     6481 caccagctcc agccatggtg tctgcactgg ctcaggcacc agcacccgtg cctgtgctgg
     6541 ctcctggacc tccacaggct gtggctccac cagcccctaa acctacacag gccggcgagg
     6601 gcacactgtc tgaagctctg ctgcagctgc agttcgacga cgaggatctg ggagccctgc
     6661 tgggaaacag caccgatcct gccgtgttca ccgacctggc cagcgtggac aacagcgagt
     6721 tccagcagct gctgaaccag ggcatccctg tggcccctca caccaccgag cccatgctga
     6781 tggaataccc cgaggccatc acccggctcg tgacaggcgc tcagaggcct cctgatccag
     6841 ctcctgcccc tctgggagca ccaggcctgc ctaatggact gctgtctggc gacgaggact
     6901 tcagctctat cgccgacatg gacttctccg cactgctggg tagcggatcg ggatctcggg
     6961 attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt agtgacgtgt
     7021 ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct ccaggaagtc
     7081 catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt ccagtacatg
     7141 agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca gcgcccgcag
     7201 tgactcccga ggccagtcac ctgttggagg atcccgatga agagacgagc caggctgtca
     7261 aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct gcaatctgtg
     7321 gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg acaaccacac
     7381 ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa ttgaacgaga
     7441 ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc agcacaggac
     7501 tgtccatctt cgacacatct ctgttttgaa agggttcgat ccctaccggt tagtaatgag
     7561 tttaaacggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     7621 atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcataaacg
     7681 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     7741 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     7801 gctcgcagcc aacgtcgggg cggcaggccc tgccatagca gatctgcgaa atcggatcca
     7861 gctaaatcta agtggatctg cgatcgctcc ggtgcccgtc agtgggcaga gcgcacatcg
     7921 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaacgggtgc ctagagaagg
     7981 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     8041 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     8101 gccgccagaa cacagctgaa gcttcgaggg gctcgcatct ctccttcacg cgcccgccgc
     8161 cctacctgag gccgccatcc acgccggttg agtcgcgttc tgccgcctcc cgcctgtggt
     8221 gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc tcaggtcgag accgggcctt
     8281 tgtccggcgc tcccttggag cctacctaga ctcagccggc tctccacgct ttgcctgacc
     8341 ctgcttgctc aactctacgt ctttgtttcg ttttctgttc tgcgccgtta cagatccaag
     8401 ctgtgaccgg cgcctactct agagccgcca tgtctagact ggacaagagc aaagtcataa
     8461 actctgctct ggaattactc aatggagtcg gtatcgaagg cctgacgaca aggaaactcg
     8521 ctcaaaagct gggagttgag cagcctaccc tgtactggca cgtgaagaac aagcgggccc
     8581 tgctcgatgc cctgccaatc gagatgctgg acaggcatca tacccactcc tgccccctgg
     8641 aaggcgagtc atggcaagac tttctgcgga acaacgccaa gtcataccgc tgtgctctcc
     8701 tctcacatcg cgacggggct aaagtgcatc tcggcacccg cccaacagag aaacagtacg
     8761 aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg cttctccctg gagaacgcac
     8821 tgtacgctct gtccgccgtg ggccacttta cactgggctg cgtattggag gaacaggagc
     8881 atcaagtagc aaaagaggaa agagagacac ctaccaccga ttctatgccc ccacttctga
     8941 aacaagcaat tgagctgttc gaccggcagg gagccgaacc tgccttcctt ttcggcctgg
     9001 aactaatcat atgtggcctg gagaaacagc taaagtgcga aagcggcggg ccgaccgacg
     9061 cccttgacga ttttgactta gacatgctcc cagccgatgc ccttgacgac tttgaccttg
     9121 atatgctgcc tgctgacgct cttgacgatt ttgaccttga catgctcccc ggggagggca
     9181 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctgaa ttcatggata
     9241 gatccggaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     9301 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     9361 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     9421 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     9481 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     9541 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     9601 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     9661 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     9721 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     9781 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     9841 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     9901 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     9961 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
    10021 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
    10081 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
    10141 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
    10201 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
    10261 aggaataggc caaggaacct ctggattaca aaatttgtga aagattgact ggtattctta
    10321 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcagttaa
    10381 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
    10441 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
    10501 tcatgtctgg aattgactca aatgatgtca attagtctat cagaagctat ctggtctccc
    10561 ttccggggga caagacatcc ctgtttaata tttaaacagc agtgttccca aactgggttc
    10621 ttatatccct tgctctggtc aaccaggttg cagggtttcc tgtcctcaca ggaacgaagt
    10681 ccctaaagaa acagtggcag ccaggtttag ccccggaatt gactggattc cttttttagg
    10741 gcccattggt atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc
    10801 gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc
    10861 acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc
    10921 gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc
    10981 tgtccctgat atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca
    11041 tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca
    11101 tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga
    11161 caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg
    11221 attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca
    11281 gactatcttt ctagggttaa tctagctgca tcaggatcat atcgtcgggt cttttttccg
    11341 gctcagtcat cgcccaagct ggcgctatct gggcatcggg gaggaagaag cccgtgcctt
    11401 ttcccgcgag gttgaagcgg catggaaaga gtttgccgag gatgactgct gctgcattga
    11461 cgttgagcga aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca tgcacgcctt
    11521 taacggtgaa ctgttcgttc aggccacctg ggataccagt tcgtcgcggc ttttccggac
    11581 acagttccgg atggtcagcc cgaagcgcat cagcaacccg aacaataccg gcgacagccg
    11641 gaactgccgt gccggtgtgc agattaatga cagcggtgcg gcgctgggat attacgtcag
    11701 cgaggacggg tatcctggct ggatgccgca gaaatggaca tggatacccc gtgagttacc
    11761 cggcgggcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
    11821 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
    11881 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
    11941 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
    12001 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
    12061 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
    12121 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
    12181 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
    12241 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
    12301 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
    12361 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
    12421 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
    12481 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
    12541 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
    12601 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
    12661 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
    12721 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
    12781 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
    12841 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
    12901 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
    12961 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
    13021 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
    13081 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
    13141 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
    13201 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
    13261 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
    13321 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
    13381 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
    13441 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
    13501 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
    13561 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
    13621 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
    13681 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
    13741 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
    13801 atactcat
//
