LOCUS       sequence-354094-       13351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac compatible plasmid expressing dCas9-VPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13351)
  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 13351)
  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: "Synthetic 
            Biology, Volume 6, Issue 1, 2021"
COMMENT     Sequence Label: NAX_CAGGS_CAS9_m4_VPR_Hygro
FEATURES             Location/Qualifiers
     source          1..13351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     546..568
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     630..649
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      718..727
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(724..750,751..753,754..3237,3238..3243,3244..3309,
                     3310..3312,3313..4827)
                     /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             4840..4860
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4885..5034
                     /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             5059..5079
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5509..5865
                     /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             5884..6453
                     /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(6478..6497)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6522..6570
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        6802..7013
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7023..7032
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7044..8063
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             7044..8063
                     /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"
     polyA_signal    8067..8188
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8104..8123)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8158..8177
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   8951..8985
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9476..9492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9476..9492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9489..9511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9500..9516
                     /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(9524..9530,9531..9548,9549..9554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9569..9590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9707..9724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9878..10466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9958..9977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10637..11428,11429..11497))
                     /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     11260..11279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11498..11602)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11628..12083)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11765..11786)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     11977..11996
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12210..12232
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12224..12241
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12225..12241
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12371..12433
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        12854..13233
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc
       61 ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc
      121 gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg
      181 gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg
      241 gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct
      301 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc
      361 gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc
      421 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
      481 ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag
      541 ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca
      601 tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca
      661 ttttggcaaa gaatctagat accctcgtaa aggtctagag ctagcgaatt cgaatttgcc
      721 accatggaca agaagtactc cattgggctc gctatcggca caaacagcgt cggctgggcc
      781 gtcattacgg acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat
      841 cgccacagca taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc
      901 gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc
      961 tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat
     1021 aggctggagg agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt
     1081 ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg
     1141 aagaagcttg tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg
     1201 catatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc
     1261 gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac
     1321 ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc
     1381 cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt
     1441 aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc
     1501 gaagatgcca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg
     1561 gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc
     1621 attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct
     1681 agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc
     1741 agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac
     1801 gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc
     1861 ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg
     1921 cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg
     1981 cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag
     2041 attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat
     2101 tccagattcg cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag
     2161 gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat
     2221 aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca
     2281 gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc
     2341 ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt
     2401 accgtgaaac agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa
     2461 atcagcggag tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa
     2521 atcattaaag acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt
     2581 gtcctcaccc ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac
     2641 gctcatctct tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg
     2701 gggcggctgt caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc
     2761 ctggattttc ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat
     2821 gactctctca cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt
     2881 cttcacgagc acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag
     2941 accgttaagg tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc
     3001 gttatcgaga tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa
     3061 aggatgaaga ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac
     3121 ccagttgaaa acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc
     3181 agggacatgt acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggct
     3241 gctatcgtgc cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga
     3301 tccgataaag ctagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg
     3361 aaaaattatt ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat
     3421 ctgactaagg ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg
     3481 cagcttgttg agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg
     3541 aacaccaagt acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag
     3601 tctaagctgg tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac
     3661 aattaccacc atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa
     3721 aaatatccca agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg
     3781 aaaatgatcg caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac
     3841 agcaatatta tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag
     3901 cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat
     3961 ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa
     4021 gtacagaccg gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg
     4081 atcgcacgca aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc
     4141 gcttacagtg tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc
     4201 gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc
     4261 gactttctcg aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc
     4321 aagtactctc tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag
     4381 ctgcagaaag gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc
     4441 agccactatg aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg
     4501 gaacaacaca aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga
     4561 gtgatcctcg ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat
     4621 aagcccatca gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc
     4681 gcgcctgcag ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca
     4741 aaggaggtcc tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga
     4801 atcgacctct ctcagctcgg tggagacagc agggctgacc ccaagaagaa gaggaaggtg
     4861 gaggccagcg gttccggacg ggctgacgca ttggacgatt ttgatctgga tatgctggga
     4921 agtgacgccc tcgatgattt tgaccttgac atgcttggtt cggatgccct tgatgacttt
     4981 gacctcgaca tgctcggcag tgacgccctt gatgatttcg acctggacat gctgattaac
     5041 tctagaagtt ccggatctcc gaaaaagaaa cgcaaagttg gtagccagta cctgcccgac
     5101 accgacgacc ggcaccggat cgaggaaaag cggaagcgga cctacgagac attcaagagc
     5161 atcatgaaga agtccccctt cagcggcccc accgacccta gacctccacc tagaagaatc
     5221 gccgtgccca gcagatccag cgccagcgtg ccaaaacctg ccccccagcc ttaccccttc
     5281 accagcagcc tgagcaccat caactacgac gagttcccta ccatggtgtt ccccagcggc
     5341 cagatctctc aggcctctgc tctggctcca gcccctcctc aggtgctgcc tcaggctcct
     5401 gctcctgcac cagctccagc catggtgtct gcactggctc aggcaccagc acccgtgcct
     5461 gtgctggctc ctggacctcc acaggctgtg gctccaccag cccctaaacc tacacaggcc
     5521 ggcgagggca cactgtctga agctctgctg cagctgcagt tcgacgacga ggatctggga
     5581 gccctgctgg gaaacagcac cgatcctgcc gtgttcaccg acctggccag cgtggacaac
     5641 agcgagttcc agcagctgct gaaccagggc atccctgtgg cccctcacac caccgagccc
     5701 atgctgatgg aataccccga ggccatcacc cggctcgtga caggcgctca gaggcctcct
     5761 gatccagctc ctgcccctct gggagcacca ggcctgccta atggactgct gtctggcgac
     5821 gaggacttca gctctatcgc cgacatggac ttctccgcac tgctgggtag cggatcggga
     5881 tctcgggatt ccagggaagg gatgtttttg ccgaagcctg aggccggctc cgctattagt
     5941 gacgtgtttg agggccgcga ggtgtgccag ccaaaacgaa tccggccatt tcatcctcca
     6001 ggaagtccat gggccaaccg cccactcccc gccagcctcg caccaacacc aaccggtcca
     6061 gtacatgagc cagtcgggtc actgaccccg gcaccagtcc ctcagccact ggatccagcg
     6121 cccgcagtga ctcccgaggc cagtcacctg ttggaggatc ccgatgaaga gacgagccag
     6181 gctgtcaaag cccttcggga gatggccgat actgtgattc cccagaagga agaggctgca
     6241 atctgtggcc aaatggacct ttcccatccg cccccaaggg gccatctgga tgagctgaca
     6301 accacacttg agtccatgac cgaggatctg aacctggact cacccctgac cccggaattg
     6361 aacgagattc tggatacctt cctgaacgac gagtgcctct tgcatgccat gcatatcagc
     6421 acaggactgt ccatcttcga cacatctctg ttttgagttt aaacggggga ggctaactga
     6481 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     6541 taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     6601 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     6661 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     6721 caggccctgc catagcagat ctgcgaaatc ggatccgcgg ccacactgat tctagctgca
     6781 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     6841 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     6901 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     6961 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggatcctc
     7021 gagccaccat ggatagatcc ggaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     7081 tgatcgaaaa gttcgacagc gtttccgacc tgatgcagct ctcggagggc gaagaatctc
     7141 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     7201 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     7261 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     7321 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     7381 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     7441 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     7501 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     7561 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     7621 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     7681 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     7741 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     7801 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     7861 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     7921 caatcgtccg atcaggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     7981 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     8041 ctcgtccgag ggcaaaggaa taggttaact tgtttattgc agcttataat ggttacaaat
     8101 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     8161 gtttgtccaa actcatcaat gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat
     8221 tagtctatca gaagctcatc tggtctccct tccgggggac aagacatccc tgtttaatat
     8281 ttaaacagca gtgttcccaa actgggttct tatatccctt gctctggtca accaggttgc
     8341 agggtttcct gtcctcacag gaacgaagtc cctaaagaaa cagtggcagc caggtttagc
     8401 cccggaattg actggattcc ttttttaggg cccattggta tggctttttc cccgtatccc
     8461 cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc
     8521 caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg
     8581 ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa
     8641 ttacatccct gggggctttg ggggggggct gtccctgata tctataacaa gaaaatatat
     8701 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
     8761 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
     8821 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
     8881 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
     8941 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctgcat
     9001 caggatcata tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg gcgctatctg
     9061 ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag
     9121 tttgccgagg atgactgctg ctgcattgac gttgagcgaa aacgcacgtt taccatgatg
     9181 attcgggaag gtgtggccat gcacgccttt aacggtgaac tgttcgttca ggccacctgg
     9241 gataccagtt cgtcgcggct tttccggaca cagttccgga tggtcagccc gaagcgcatc
     9301 agcaacccga acaataccgg cgacagccgg aactgccgtg ccggtgtgca gattaatgac
     9361 agcggtgcgg cgctgggata ttacgtcagc gaggacgggt atcctggctg gatgccgcag
     9421 aaatggacat ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat
     9481 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     9541 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     9601 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     9661 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     9721 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     9781 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     9841 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     9901 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     9961 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
    10021 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
    10081 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
    10141 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
    10201 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
    10261 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
    10321 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
    10381 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    10441 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    10501 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    10561 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    10621 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    10681 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    10741 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    10801 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    10861 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    10921 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    10981 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    11041 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    11101 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    11161 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    11221 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    11281 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    11341 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    11401 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    11461 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    11521 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    11581 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
    11641 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
    11701 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
    11761 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
    11821 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
    11881 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
    11941 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
    12001 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
    12061 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
    12121 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
    12181 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
    12241 tgagcgcgcc tcgttcattc acgtttttga acccgtggag gacgggcaga ctcgcggtgc
    12301 aaatgtgttt tacagcgtga tggagcagat gaagatgctc gacacgctgc agaacacgca
    12361 gctagattaa ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgtgta
    12421 aaattgacgc atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga
    12481 tattaagttt tattatattt acacttacat actaataata aattcaacaa acaatttatt
    12541 tatgtttatt tatttattaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa
    12601 acttttatga gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc
    12661 gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc
    12721 cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt
    12781 cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa
    12841 ggcctccacg gccgacattg attattgact agttattaat agtaatcaat tacggggtca
    12901 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
    12961 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
    13021 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
    13081 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
    13141 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
    13201 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
    13261 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
    13321 ttttgtgcag cgatgggggc gggggggggg g
//
