LOCUS       sequence-192399-       12343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes for Cas9-VP64,  MS2-p65-HSF1, mCherry and for the gRNA 2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12343)
  AUTHORS   Fidanza A, Lopez-Yrigoyen M, Romano N, Jones R, Taylor AH, Forrester
            LM
  TITLE     An all-in-one UniSam vector system for efficient gene activation.
  JOURNAL   Sci Rep. 2017 Jul 25;7(1):6394. doi: 10.1038/s41598-017-06468-6.
  PUBMED    28743878
REFERENCE   2  (bases 1 to 12343)
  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: "Sci Rep."; 
            date: "2017-07-25"; volume: "7(1):6394. doi"; pages: " 
            10.1038/s41598-017-06468-6"
COMMENT     Sequence Label: PB-UniSAM
FEATURES             Location/Qualifiers
     source          1..12343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        366..1544
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          597..1535
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1492..1512
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1563..1583
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1593..1616,1617..1619,1620..4106,4107..4109,
                     4110..5693)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1593..5693
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5748..5768
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5793..5942
                     /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             5961..6017
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6030..6416
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             6471..6491
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6507..7049
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             7074..7445
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             7461..7514
                     /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             7515..8225
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMSFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(7660..7678)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    8242..8290
                     /note="synthetic polyadenylation signal"
     promoter        8353..8601
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8353..8373
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8524..8543
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8637..8655
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        8707..8725
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     repeat_region   9144..9178
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(9327..9345)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9366..9382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9366..9382)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9379..9401)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9390..9406
                     /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(9414..9420,9421..9438,9439..9444))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9459..9480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9597..9614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9768..10356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9848..9867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10527..11318,11319..11387))
                     /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     11150..11169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11388..11492)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11518..11973)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11655..11676)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     11867..11886
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12100..12122
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12114..12131
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12115..12131
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12138..12157
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        12138..12156
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   12223..12285
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 attatattta cacttacata ctaataataa attcaacaaa caatttattt atgtttattt
       61 atttattaaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa acttttaaac
      121 attctctctt ttacaaaaat aaacttattt tgtactttaa aaacagtcat gttgtattat
      181 aaaataagta attagcttaa cctatacata atagaaacaa attatactta ttagtcagtc
      241 agaaacaact ttggcacata tcaatattat gctctcgtta attaatgcaa agatggataa
      301 agttttaaac agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg
      361 gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
      421 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
      481 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
      541 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
      601 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
      661 gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg
      721 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
      781 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg
      841 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt
      901 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
      961 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc
     1021 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
     1081 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg
     1141 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat
     1201 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct
     1261 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc
     1321 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
     1381 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga
     1441 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc
     1501 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgacgtacg gccaccatga
     1561 gccccaagaa gaagagaaag gtggaggcca gcgacaagaa gtacagcatc ggcctggcca
     1621 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     1681 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     1741 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     1801 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     1861 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     1921 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     1981 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     2041 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     2101 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     2161 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     2221 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     2281 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     2341 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     2401 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     2461 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     2521 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     2581 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     2641 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     2701 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     2761 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     2821 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     2881 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     2941 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     3001 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     3061 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     3121 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     3181 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     3241 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     3301 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     3361 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     3421 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     3481 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     3541 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     3601 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     3661 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     3721 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     3781 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     3841 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     3901 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     3961 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     4021 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     4081 accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg aaggacgact
     4141 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     4201 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     4261 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     4321 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     4381 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     4441 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     4501 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     4561 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     4621 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     4681 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     4741 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     4801 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     4861 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     4921 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     4981 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     5041 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     5101 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     5161 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     5221 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     5281 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     5341 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     5401 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     5461 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     5521 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     5581 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     5641 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacagcgctg
     5701 gaggaggtgg aagcggagga ggaggaagcg gaggaggagg tagcggacct aagaaaaaga
     5761 ggaaggtggc ggccgctgga tccggacggg ctgacgcatt ggacgatttt gatctggata
     5821 tgctgggaag tgacgccctc gatgattttg accttgacat gcttggttcg gatgcccttg
     5881 atgactttga cctcgacatg ctcggcagtg acgcccttga tgatttcgac ctggacatgc
     5941 tgattaacgg aggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg
     6001 aggagaaccc tggacctgga agcggaatgg cttcaaactt tactcagttc gtgctcgtgg
     6061 acaatggtgg gacaggggat gtgacagtgg ctccttctaa tttcgctaat ggggtggcag
     6121 agtggatcag ctccaactca cggagccagg cctacaaggt gacatgcagc gtcaggcagt
     6181 ctagtgccca gaagagaaag tataccatca aggtggaggt ccccaaagtg gctacccaga
     6241 cagtgggcgg agtcgaactg cctgtcgccg cttggaggtc ctacctgaac atggagctca
     6301 ctatcccaat tttcgctacc aattctgact gtgaactcat cgtgaaggca atgcaggggc
     6361 tcctcaaaga cggtaatcct atcccttccg ccatcgccgc taactcaggt atctacagcg
     6421 ctggaggagg tggaagcgga ggaggaggaa gcggaggagg aggtagcgga cctaagaaaa
     6481 agaggaaggt ggcggccgct ggatcccctt cagggcagat cagcaaccag gccctggctc
     6541 tggcccctag ctccgctcca gtgctggccc agactatggt gccctctagt gctatggtgc
     6601 ctctggccca gccacctgct ccagcccctg tgctgacccc aggaccaccc cagtcactga
     6661 gcgctccagt gcccaagtct acacaggccg gcgaggggac tctgagtgaa gctctgctgc
     6721 acctgcagtt cgacgctgat gaggacctgg gagctctgct ggggaacagc accgatcccg
     6781 gagtgttcac agatctggcc tccgtggaca actctgagtt tcagcagctg ctgaatcagg
     6841 gcgtgtccat gtctcatagt acagccgaac caatgctgat ggagtacccc gaagccatta
     6901 cccggctggt gaccggcagc cagcggcccc ccgaccccgc tccaactccc ctgggaacca
     6961 gcggcctgcc taatgggctg tccggagatg aggacttctc aagcatcgct gatatggact
     7021 ttagtgccct gctgtcacag atttcctcta gtgggcaggg aggaggtgga agcggcttca
     7081 gcgtggacac cagtgccctg ctggacctgt tcagcccctc ggtgaccgtg cccgacatga
     7141 gcctgcctga ccttgacagc agcctggcca gtatccaaga gctcctgtct ccccaggagc
     7201 cccccaggcc tcccgaggca gagaacagca gcccggattc agggaagcag ctggtgcact
     7261 acacagcgca gccgctgttc ctgctggacc ccggctccgt ggacaccggg agcaacgacc
     7321 tgccggtgct gtttgagctg ggagagggct cctacttctc cgaaggggac ggcttcgccg
     7381 aggaccccac catctccctg ctgacaggct cggagcctcc caaagccaag gaccccactg
     7441 tctccgctag cggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg
     7501 agaatcctgg cccaatggtg agcaagggcg aggaggataa catggccatc atcaaggagt
     7561 tcatgagctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     7621 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     7681 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     7741 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     7801 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     7861 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     7921 ccgacggccc cgtaatgcag aagaaaacca tgggctggga ggcctcctcc gagcggatgt
     7981 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     8041 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     8101 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     8161 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     8221 agtagtgata aaagcggccg caataaaata tctttatttt cattacatct gtgtgttggt
     8281 tttttgtgtg aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     8341 tgctcagcta gcgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     8401 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     8461 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     8521 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     8581 ttgtggaaag gacgaaacac cgggtcttcg agaagacctg ttttagagct aggccaacat
     8641 gaggatcacc catgtctgca gggcctagca agttaaaata aggctagtcc gttatcaact
     8701 tggccaacat gaggatcacc catgtctgca gggccaagtg gcaccgagtc ggtgcttttt
     8761 ttgaattctt taattaagct agccaacaag ctcgtcatcg ctttgcagaa gagcagagag
     8821 gatatgctca tcgtctaaag aactacccat tttattatat attagtcacg atatctataa
     8881 caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt
     8941 atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac
     9001 gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga
     9061 gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa
     9121 gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt
     9181 aaaaaagatt tgcgctttac tcgacctaaa ctttaaacac gtcatagaat cttcgtttga
     9241 caaaaaccac attgtggcca agctgtgtga cgcgacgcgc gctaaagaat ggcaaaccaa
     9301 gtcgcgcgag gtaccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa
     9361 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     9421 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     9481 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     9541 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     9601 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     9661 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     9721 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     9781 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     9841 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     9901 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     9961 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
    10021 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
    10081 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
    10141 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
    10201 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
    10261 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
    10321 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
    10381 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
    10441 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
    10501 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
    10561 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
    10621 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
    10681 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
    10741 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
    10801 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
    10861 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
    10921 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
    10981 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
    11041 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
    11101 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
    11161 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
    11221 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
    11281 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
    11341 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
    11401 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
    11461 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
    11521 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
    11581 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
    11641 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
    11701 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
    11761 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
    11821 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
    11881 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
    11941 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
    12001 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
    12061 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
    12121 cgacggccag tgaattgtaa tacgactcac tatagggcga attggagctc ggtattcacg
    12181 acagcaggct gaataataaa aaaattagaa actattattt aaccctagaa agataatcat
    12241 attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt tatcggtctg
    12301 tatatcgagg tttatttatt aatttgaata gatattaagt ttt
//
