LOCUS       sequence-390323-       12586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CRISPRa components including dCas9-VP64, Synergistic 
            Activation Mediators (SAM), mCherry, and gRNA expression cassette, 
            blasticidin selective .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12586)
  AUTHORS   Melo EP, Konno T, Farace I, Awadelkareem MA, Skov LR, Teodoro F, 
            Sancho TP, Paton AW, Paton JC, Fares M, Paulo PMR, Zhang X, Avezov E
  TITLE     Stress-induced protein disaggregation in the endoplasmic reticulum 
            catalysed by BiP.
  JOURNAL   Nat Commun. 2022 May 6;13(1):2501. doi: 10.1038/s41467-022-30238-2.
  PUBMED    35523806
REFERENCE   2  (bases 1 to 12586)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2022-05-6"; volume: "13(1):2501. doi"; pages: " 
            10.1038/s41467-022-30238-2"
COMMENT     Sequence Label: pCRISPRa_all-in-one
FEATURES             Location/Qualifiers
     source          1..12586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1186..1195
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1195..1581
                     /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             1636..1656
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1672..2214
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             2239..2610
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             2626..2679
                     /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             2680..3384
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2822..2840)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3090..3109
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3394..3447
                     /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             3448..3495
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(3496..3519,3520..3522,3523..6078,6079..6081,
                     6082..7596)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7651..7671
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7696..7845
                     /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             7867..7920
                     /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             7921..8319
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(8663..8680)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8669..8893
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   9180..9214
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9234..9254)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9234..9252)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9273..9289)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9273..9289)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9286..9308)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9297..9313
                     /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(9321..9327,9328..9345,9346..9351))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9366..9387
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9504..9521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9675..10263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9755..9774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10434..11225,11226..11294))
                     /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     11057..11076
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11295..11399)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11425..11880)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11562..11583)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     11774..11793
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        12022..12270
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     12022..12042
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     12193..12212
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        12320..12338
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        12390..12408
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatgaagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgac gtacggccac catggcttca
     1201 aactttactc agttcgtgct cgtggacaat ggtgggacag gggatgtgac agtggctcct
     1261 tctaatttcg ctaatggggt ggcagagtgg atcagctcca actcacggag ccaggcctac
     1321 aaggtgacat gcagcgtcag gcagtctagt gcccagaaga gaaagtatac catcaaggtg
     1381 gaggtcccca aagtggctac ccagacagtg ggcggagtcg aactgcctgt cgccgcttgg
     1441 aggtcctacc tgaacatgga gctcactatc ccaattttcg ctaccaattc tgactgtgaa
     1501 ctcatcgtga aggcaatgca ggggctcctc aaagacggta atcctatccc ttccgccatc
     1561 gccgctaact caggtatcta cagcgctgga ggaggtggaa gcggaggagg aggaagcgga
     1621 ggaggaggta gcggacctaa gaaaaagagg aaggtggcgg ccgctggatc cccttcaggg
     1681 cagatcagca accaggccct ggctctggcc cctagctccg ctccagtgct ggcccagact
     1741 atggtgccct ctagtgctat ggtgcctctg gcccagccac ctgctccagc ccctgtgctg
     1801 accccaggac caccccagtc actgagcgct ccagtgccca agtctacaca ggccggcgag
     1861 gggactctga gtgaagctct gctgcacctg cagttcgacg ctgatgagga cctgggagct
     1921 ctgctgggga acagcaccga tcccggagtg ttcacagatc tggcctccgt ggacaactct
     1981 gagtttcagc agctgctgaa tcagggcgtg tccatgtctc atagtacagc cgaaccaatg
     2041 ctgatggagt accccgaagc cattacccgg ctggtgaccg gcagccagcg gccccccgac
     2101 cccgctccaa ctcccctggg aaccagcggc ctgcctaatg ggctgtccgg agatgaagac
     2161 ttctcaagca tcgctgatat ggactttagt gccctgctgt cacagatttc ctctagtggg
     2221 cagggaggag gtggaagcgg cttcagcgtg gacaccagtg ccctgctgga cctgttcagc
     2281 ccctcggtga ccgtgcccga catgagcctg cctgaccttg acagcagcct ggccagtatc
     2341 caagagctcc tgtctcccca ggagcccccc aggcctcccg aggcagagaa cagcagcccg
     2401 gattcaggga agcagctggt gcactacaca gcgcagccgc tgttcctgct ggaccccggc
     2461 tccgtggaca ccgggagcaa cgacctgccg gtgctgtttg agctgggaga gggctcctac
     2521 ttctccgaag gggacggctt cgccgaggac cccaccatct ccctgctgac aggctcggag
     2581 cctcccaaag ccaaggaccc cactgtctcc tgtacaggca gtggagaggg cagaggaagt
     2641 ctgctaacat gcggtgacgt cgaggagaat cctggcccag tgagcaaggg cgaggaggat
     2701 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     2761 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     2821 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     2881 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     2941 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     3001 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     3061 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     3121 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     3181 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     3241 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     3301 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     3361 ggcggcatgg acgagctgta caagggatcc ggagagggca gaggaagtct cctaacatgc
     3421 ggtgacgtgg aggagaatcc tggcccaaaa aggccggcgg ccacgaaaaa ggccggccag
     3481 gcaaaaaaga aaaaggacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg
     3541 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
     3601 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
     3661 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
     3721 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
     3781 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
     3841 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
     3901 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     3961 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     4021 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     4081 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     4141 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     4201 ctgttcggca acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     4261 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     4321 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     4381 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     4441 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     4501 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     4561 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     4621 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     4681 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     4741 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     4801 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     4861 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     4921 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     4981 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     5041 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     5101 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     5161 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     5221 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     5281 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     5341 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     5401 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     5461 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     5521 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     5581 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     5641 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     5701 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     5761 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     5821 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     5881 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     5941 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     6001 gatgtggacc acatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     6061 ctgaccagaa gcgacaaggc ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     6121 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     6181 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     6241 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     6301 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     6361 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     6421 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     6481 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     6541 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     6601 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     6661 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     6721 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     6781 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     6841 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     6901 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     6961 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     7021 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     7081 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     7141 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     7201 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     7261 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     7321 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     7381 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     7441 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     7501 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     7561 gagacacgga tcgacctgtc tcagctggga ggcgacagcg ctggaggagg tggaagcgga
     7621 ggaggaggaa gcggaggagg aggtagcgga cctaagaaaa agaggaaggt ggcggccgct
     7681 ggatccggac gggctgacgc attggacgat tttgatctgg atatgctggg aagtgacgcc
     7741 ctcgatgatt ttgaccttga catgcttggt tcggatgccc ttgatgactt tgacctcgac
     7801 atgctcggca gtgacgccct tgatgatttc gacctggaca tgctgattaa ctgtacaggc
     7861 agtggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca
     7921 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
     7981 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
     8041 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
     8101 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
     8161 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
     8221 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
     8281 cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag tacctttaag accaatgact
     8341 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     8401 attcactccc aaagaagtca agatctgctt tttgcctgta ctgggtctct ctggttagac
     8461 cagagtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     8521 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     8581 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     8641 gcagtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     8701 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     8761 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     8821 gtggggcagg acagcaaggg ggaggattgg gaagtcaata gcaggcatgc tggggatgcg
     8881 gtgggctcta tgggcggccg ttaatgatat ctataacaag aaaatatata tataataagt
     8941 tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag
     9001 tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag
     9061 tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc
     9121 ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc
     9181 atgcgtcaat tttacgcaga ctatctttct agggttaaat taagcttttg ttccctttag
     9241 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     9301 tatccgctca caattccaca caacatacga gccgggagca taaagtgtaa agcctggggt
     9361 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     9421 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     9481 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     9541 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     9601 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     9661 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     9721 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     9781 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     9841 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     9901 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     9961 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
    10021 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
    10081 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
    10141 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
    10201 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
    10261 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
    10321 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
    10381 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
    10441 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
    10501 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
    10561 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
    10621 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
    10681 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
    10741 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
    10801 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
    10861 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
    10921 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
    10981 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
    11041 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
    11101 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
    11161 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
    11221 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
    11281 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
    11341 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
    11401 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
    11461 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
    11521 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
    11581 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
    11641 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
    11701 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
    11761 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
    11821 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
    11881 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
    11941 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
    12001 ggttttccca gtcacgacgt tgagggccta tttcccatga ttccttcata tttgcatata
    12061 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
    12121 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
    12181 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
    12241 ttatatatct tgtggaaagg acgaaacacc ggagacgtta tattccaaag tgtaaacgtc
    12301 tcgttttaga gctaggccaa catgaggatc acccatgtct gcagggccta gcaagttaaa
    12361 ataaggctag tccgttatca acttggccaa catgaggatc acccatgtct gcagggccaa
    12421 gtggcaccga gtcggtgctt tttttgcttt gttactttat agaagaaatt ttgagttttt
    12481 gttttttttt aataaataaa taaacataaa taaattgttt gttgaattta ttattagtat
    12541 gtaagtgtaa atataataaa acttaatatc tattcaaatt aataaa
//
