LOCUS       sequence-373705-       11406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 fused with tagRFPt, P2A site and tagBFP, expressed under 
            EF1-alpha promoter in a piggyBac plasmid. Expresses Blasticidin 
            resistance under PGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11406)
  AUTHORS   Noviello G, Gjaltema RAF, Schulz EG
  TITLE     CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning 
            of endogenous gene expression.
  JOURNAL   Nat Commun. 2023 Jun 3;14(1):3225. doi: 10.1038/s41467-023-38909-4.
  PUBMED    37270532
REFERENCE   2  (bases 1 to 11406)
  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: "2023-06-3"; volume: "14(1):3225. doi"; pages: " 
            10.1038/s41467-023-38909-4"
COMMENT     Sequence Label: pSLPB2-SpdCas9-tagRFPt-P2A-tagBFP-PGK-Blasticidin
FEATURES             Location/Qualifiers
     source          1..11406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             29..49
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(50..73,74..76,77..2563,2564..2566,2567..4150)
                     /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             50..4150
                     /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             4151..4171
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4172..4189
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4244..4972
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     CDS             4991..5047
                     /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             5048..5749
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    5764..5988
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6339..6358
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6414..6812
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    6813..7401
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6866..6886)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7284..7295)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   7584..7646
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7676..7696)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7676..7694)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7715..7731)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7715..7731)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7728..7750)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7739..7755
                     /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(7763..7769,7770..7787,7788..7793))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7808..7829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7946..7963)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8117..8705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8197..8216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8876..9667,9668..9736))
                     /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     9499..9518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9737..9841)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(9867..10322)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10004..10025)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10216..10235
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10449..10471
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     10463..10480
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     10464..10480
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10490..10509
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        10490..10508
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   10524..10558
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        10880..11091
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             11104..11372
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
ORIGIN
        1 aattcagagc ggccgcgccg ccaccatgcc taagaaaaag aggaaggtgg acaagaagta
       61 cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
      121 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
      181 gaacctgatc ggcgccctgc tgttcgacag cggagaaaca gccgaggcca cccggctgaa
      241 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
      301 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
      361 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
      421 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
      481 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
      541 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
      601 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
      661 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
      721 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag
      781 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
      841 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
      901 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
      961 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     1021 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     1081 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acatcgatgg
     1141 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     1201 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     1261 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     1321 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     1381 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     1441 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     1501 cgccagcgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     1561 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtaca acgagctgac
     1621 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     1681 aaaagccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     1741 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     1801 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     1861 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     1921 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     1981 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     2041 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     2101 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     2161 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     2221 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     2281 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     2341 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     2401 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     2461 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     2521 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gacgctatcg tgcctcagag
     2581 ctttctgaag gacgactcca tcgataacaa agtgctgact cggagcgaca agaaccgggg
     2641 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcgcca
     2701 gctgctgaat gccaagctga ttacccagag gaagttcgac aatctgacca aggccgagag
     2761 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     2821 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     2881 gaacgacaaa ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     2941 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     3001 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     3061 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     3121 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     3181 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     3241 aaacggcgaa acaggcgaga tcgtgtggga taagggccgg gactttgcca ccgtgcggaa
     3301 agtgctgtct atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     3361 cagcaaagag tctatcctgc ccaagaggaa cagcgacaag ctgatcgcca gaaagaagga
     3421 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     3481 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     3541 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     3601 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     3661 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     3721 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     3781 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaaacacta
     3841 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     3901 taatctggac aaggtgctga gcgcctacaa caagcacaga gacaagccta tcagagagca
     3961 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     4021 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     4081 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     4141 gggaggcgac cccaagaaga agcgcaaggt gcaccaccac caccaccacg gaggaggtgg
     4201 aagcggagga ggaggaagcg gaggaggagg tagctggcgc gccgtgtcta agggcgaaga
     4261 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca
     4321 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat
     4381 caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat
     4441 gtacggcagc agaaccttca tcaaccacac ccagggcatc cccgacttct ttaagcagtc
     4501 cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac
     4561 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg
     4621 ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggccaa
     4681 caccgagatg ctgtaccccg ctgacggcgg cctggaaggc agaaccgaca tggccctgaa
     4741 gctcgtgggc gggggccacc tgatctgcaa cttcaagacc acatacagat ccaagaaacc
     4801 cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac cacagactgg aaagaatcaa
     4861 ggaggccgac aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga
     4921 cctccctagc aaactggggc acaaacttaa tggcatggac gagctgtaca agttaattaa
     4981 cggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc
     5041 tggacctatg agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     5101 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     5161 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     5221 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
     5281 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     5341 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     5401 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     5461 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     5521 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata
     5581 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     5641 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     5701 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaga cgcgtattaa
     5761 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     5821 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     5881 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     5941 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggcc tctggcctcg
     6001 cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc
     6061 gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg
     6121 caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc
     6181 tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc
     6241 tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag
     6301 gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg
     6361 cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct gcaatggcca
     6421 agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca
     6481 tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct
     6541 tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg
     6601 gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga
     6661 acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg
     6721 ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat
     6781 tgctgccctc tggttatgtg tgggagggct aaaatcaacc tctggattac aaaatttgtg
     6841 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     6901 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     6961 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     7021 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     7081 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     7141 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     7201 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     7261 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     7321 tgctgccggc tctgcggcct cttccgcgtc tccgccttcg ccctcagacg agtcggatct
     7381 ccctttgggc cgcctccccg ccggtaccta aaagttttgt tactttatag aagaaatttt
     7441 gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt
     7501 attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc
     7561 tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc tttaacgtac
     7621 gtcacaatat gattatcttt ctagggttaa tctagtatta atccagcttt tgttcccttt
     7681 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     7741 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     7801 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     7861 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     7921 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     7981 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     8041 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     8101 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     8161 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     8221 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     8281 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     8341 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     8401 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     8461 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     8521 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     8581 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     8641 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     8701 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     8761 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     8821 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     8881 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     8941 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     9001 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     9061 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     9121 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     9181 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     9241 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     9301 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     9361 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     9421 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     9481 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     9541 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     9601 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     9661 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     9721 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     9781 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     9841 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     9901 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     9961 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
    10021 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
    10081 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
    10141 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
    10201 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
    10261 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
    10321 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
    10381 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
    10441 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
    10501 actatagggc gaattggggc taaccctaga aagatagtct gcgtaaaatt gacgcatgca
    10561 ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac
    10621 atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg
    10681 attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt
    10741 ttaagattta actcatacga taattatatt gttatttcat gttctactta cgtgataact
    10801 tattatatat atattttctt gttatagata tcaactagaa tacgcgtaag gatctgcgat
    10861 cgctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
    10921 ggaggggtcg gcaattgaac gggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
    10981 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
    11041 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca gctgaagctt
    11101 cgaggggctc gcatctctcc ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc
    11161 cggttgagtc gcgttctgcc gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc
    11221 taggtaagtt taaagctcag gtcgagaccg ggcctttgtc cggcgctccc ttggagccta
    11281 cctagactca gccggctctc cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt
    11341 gtttcgtttt ctgttctgcg ccgttacaga tccaagctgt gaccggcgcc tactctagag
    11401 ctagcg
//
