LOCUS       sequence-354093-       13535 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac compatible plasmid expressing dCas9-VPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13535)
  AUTHORS   Nghi Dang , Alissa Lance-Byrne, Angela Tung, Xiaoge Guo, Ryan J 
            Cecchi, Joanna Buchthal, Alejandro Chavez, Nan Cher Yeo
  TITLE     Generation and application of a versatile CRISPR toolkit for 
            mammalian cell engineering
  JOURNAL   Synthetic Biology, Volume 6, Issue 1, 2021
REFERENCE   2  (bases 1 to 13535)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Synthetic 
            Biology, Volume 6, Issue 1, 2021"
COMMENT     Sequence Label: NAX_CAGGS_CAS9_m4_VPR_EYFP
FEATURES             Location/Qualifiers
     source          1..13535
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          110..1126
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1050..1072
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1134..1153
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1222..1231
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1228..1254,1255..1257,1258..3741,3742..3747,3748..3813,
                     3814..3816,3817..5331)
                     /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             5344..5364
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5389..5538
                     /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             5563..5583
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6013..6369
                     /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             6388..6957
                     /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(6982..7001)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7026..7074
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7306..7517
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7527..7536
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7533..7535,7536..7538,7539..8252)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7578..7599)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7839..7858)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8186..8207
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    8256..8377
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8293..8312)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8347..8366
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9140..9174
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9665..9681)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9665..9681)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9678..9700)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9689..9705
                     /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(9713..9719,9720..9737,9738..9743))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9758..9779
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9896..9913)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10067..10655)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10147..10166)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10826..11617,11618..11686))
                     /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     11449..11468
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11687..11791)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11817..12272)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11954..11975)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12166..12185
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12399..12421
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12413..12430
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12414..12430
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12560..12622
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        13043..13422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
       61 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
      121 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
      181 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
      241 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
      301 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
      361 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
      421 cgcggggctt tttgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
      481 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
      541 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
      601 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
      661 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
      721 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg
      781 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
      841 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
      901 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
      961 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
     1021 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     1081 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     1141 gctggttatt gtgctgtctc atcattttgg caaagaatct agataccctc gtaaaggtct
     1201 agagctagcg aattcgaatt tgccaccatg gacaagaagt actccattgg gctcgctatc
     1261 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
     1321 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
     1381 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
     1441 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     1501 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     1561 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     1621 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     1681 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     1741 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     1801 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     1861 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     1921 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     1981 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     2041 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     2101 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     2161 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     2221 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     2281 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     2341 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     2401 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     2461 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     2521 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     2581 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     2641 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     2701 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     2761 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     2821 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     2881 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     2941 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     3001 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     3061 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     3121 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     3181 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     3241 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     3301 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     3361 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     3421 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     3481 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     3541 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     3601 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     3661 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     3721 cggctctccg actacgacgt ggctgctatc gtgccccagt cttttctcaa agatgattct
     3781 attgataata aagtgttgac aagatccgat aaagctagag ggaagagtga taacgtcccc
     3841 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     3901 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     3961 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     4021 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     4081 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     4141 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     4201 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     4261 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     4321 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     4381 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     4441 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     4501 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     4561 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     4621 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     4681 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     4741 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     4801 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     4861 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     4921 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     4981 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     5041 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     5101 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     5161 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     5221 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     5281 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     5341 gaccccaaga agaagaggaa ggtggaggcc agcggttccg gacgggctga cgcattggac
     5401 gattttgatc tggatatgct gggaagtgac gccctcgatg attttgacct tgacatgctt
     5461 ggttcggatg cccttgatga ctttgacctc gacatgctcg gcagtgacgc ccttgatgat
     5521 ttcgacctgg acatgctgat taactctaga agttccggat ctccgaaaaa gaaacgcaaa
     5581 gttggtagcc agtacctgcc cgacaccgac gaccggcacc ggatcgagga aaagcggaag
     5641 cggacctacg agacattcaa gagcatcatg aagaagtccc ccttcagcgg ccccaccgac
     5701 cctagacctc cacctagaag aatcgccgtg cccagcagat ccagcgccag cgtgccaaaa
     5761 cctgcccccc agccttaccc cttcaccagc agcctgagca ccatcaacta cgacgagttc
     5821 cctaccatgg tgttccccag cggccagatc tctcaggcct ctgctctggc tccagcccct
     5881 cctcaggtgc tgcctcaggc tcctgctcct gcaccagctc cagccatggt gtctgcactg
     5941 gctcaggcac cagcacccgt gcctgtgctg gctcctggac ctccacaggc tgtggctcca
     6001 ccagccccta aacctacaca ggccggcgag ggcacactgt ctgaagctct gctgcagctg
     6061 cagttcgacg acgaggatct gggagccctg ctgggaaaca gcaccgatcc tgccgtgttc
     6121 accgacctgg ccagcgtgga caacagcgag ttccagcagc tgctgaacca gggcatccct
     6181 gtggcccctc acaccaccga gcccatgctg atggaatacc ccgaggccat cacccggctc
     6241 gtgacaggcg ctcagaggcc tcctgatcca gctcctgccc ctctgggagc accaggcctg
     6301 cctaatggac tgctgtctgg cgacgaggac ttcagctcta tcgccgacat ggacttctcc
     6361 gcactgctgg gtagcggatc gggatctcgg gattccaggg aagggatgtt tttgccgaag
     6421 cctgaggccg gctccgctat tagtgacgtg tttgagggcc gcgaggtgtg ccagccaaaa
     6481 cgaatccggc catttcatcc tccaggaagt ccatgggcca accgcccact ccccgccagc
     6541 ctcgcaccaa caccaaccgg tccagtacat gagccagtcg ggtcactgac cccggcacca
     6601 gtccctcagc cactggatcc agcgcccgca gtgactcccg aggccagtca cctgttggag
     6661 gatcccgatg aagagacgag ccaggctgtc aaagcccttc gggagatggc cgatactgtg
     6721 attccccaga aggaagaggc tgcaatctgt ggccaaatgg acctttccca tccgccccca
     6781 aggggccatc tggatgagct gacaaccaca cttgagtcca tgaccgagga tctgaacctg
     6841 gactcacccc tgaccccgga attgaacgag attctggata ccttcctgaa cgacgagtgc
     6901 ctcttgcatg ccatgcatat cagcacagga ctgtccatct tcgacacatc tctgttttga
     6961 gtttaaacgg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     7021 tatgacggca ataaaaagac agaataaaac gcacgggtgt tgggtcgttt gttcataaac
     7081 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     7141 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     7201 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc agatctgcga aatcggatcc
     7261 gcggccgcac tgattctagc tgcagaggct ccggtgcccg tcagtgggca gagcgcacat
     7321 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
     7381 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     7441 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     7501 ttgccgccag aacacaggat cctcgagcca ccatggtgag caagggcgag gagctgttca
     7561 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     7621 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     7681 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcctgc
     7741 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     7801 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     7861 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     7921 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     7981 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     8041 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     8101 gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc aagctgagca
     8161 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     8221 tcactctcgg catggacgag ctgtacaagt aagttaactt gtttattgca gcttataatg
     8281 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     8341 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggaat tgactcaaat
     8401 gatgtctatt agtctatcag aagctcatct ggtctccctt ccgggggaca agacatccct
     8461 gtttaatatt taaacagcag tgttcccaaa ctgggttctt atatcccttg ctctggtcaa
     8521 ccaggttgca gggtttcctg tcctcacagg aacgaagtcc ctaaagaaac agtggcagcc
     8581 aggtttagcc ccggaattga ctggattcct tttttagggc ccattggtat ggctttttcc
     8641 ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg
     8701 atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg
     8761 gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga
     8821 gggacgtaat tacatccctg ggggctttgg gggggggctg tccctgatat ctataacaag
     8881 aaaatatata tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg
     8941 tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg
     9001 tcgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc
     9061 caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga
     9121 gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaatc
     9181 tagctgcatc aggatcatat cgtcgggtct tttttccggc tcagtcatcg cccaagctgg
     9241 cgctatctgg gcatcgggga ggaagaagcc cgtgcctttt cccgcgaggt tgaagcggca
     9301 tggaaagagt ttgccgagga tgactgctgc tgcattgacg ttgagcgaaa acgcacgttt
     9361 accatgatga ttcgggaagg tgtggccatg cacgccttta acggtgaact gttcgttcag
     9421 gccacctggg ataccagttc gtcgcggctt ttccggacac agttccggat ggtcagcccg
     9481 aagcgcatca gcaacccgaa caataccggc gacagccgga actgccgtgc cggtgtgcag
     9541 attaatgaca gcggtgcggc gctgggatat tacgtcagcg aggacgggta tcctggctgg
     9601 atgccgcaga aatggacatg gataccccgt gagttacccg gcgggcgcgc ttggcgtaat
     9661 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     9721 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     9781 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     9841 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     9901 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     9961 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
    10021 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
    10081 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
    10141 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
    10201 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
    10261 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
    10321 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
    10381 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
    10441 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
    10501 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
    10561 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
    10621 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
    10681 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
    10741 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
    10801 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
    10861 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
    10921 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
    10981 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
    11041 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
    11101 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
    11161 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
    11221 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
    11281 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
    11341 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
    11401 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
    11461 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
    11521 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
    11581 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
    11641 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
    11701 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
    11761 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
    11821 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
    11881 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
    11941 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
    12001 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
    12061 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
    12121 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
    12181 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
    12241 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
    12301 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
    12361 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
    12421 gacggccagt gagcgcgcct cgttcattca cgtttttgaa cccgtggagg acgggcagac
    12481 tcgcggtgca aatgtgtttt acagcgtgat ggagcagatg aagatgctcg acacgctgca
    12541 gaacacgcag ctagattaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa
    12601 tcatgtgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat
    12661 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa
    12721 caatttattt atgtttattt atttattaaa aaaaacaaaa actcaaaatt tcttctataa
    12781 agtaacaaaa cttttatgag ggacagcccc cccccaaagc ccccagggat gtaattacgt
    12841 ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc
    12901 ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg
    12961 gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tggggggata
    13021 cggggaaaag gcctccacgg ccgacattga ttattgacta gttattaata gtaatcaatt
    13081 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
    13141 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
    13201 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
    13261 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
    13321 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
    13381 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
    13441 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
    13501 ttttaattat tttgtgcagc gatgggggcg ggggg
//
