LOCUS       sequence-373701-       12190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ER50 degron-tagged 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 12190)
  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 12190)
  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: 
            pSLPB2B-ER50-SpdCas9-tagRFPt-P2A-tagBFP-PGK-Blasticidin
FEATURES             Location/Qualifiers
     source          1..12190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     412..431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(650..754)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(780..1235)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(917..938)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1129..1148
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1362..1384
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1376..1393
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1377..1393
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1403..1422
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1403..1421
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1437..1471
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1793..2004
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2017..2285
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2337..2357
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3153..3176,3177..3179,3180..5666,5667..5669,
                     5670..7253)
                     /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             3153..7253
                     /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             7254..7274
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7275..7292
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7347..8075
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     CDS             8094..8150
                     /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             8151..8852
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    8867..9091
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     9442..9461
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             9517..9915
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9916..10504
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9969..9989)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10387..10398)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   10687..10749
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10779..10799)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10779..10797)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10818..10834)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10818..10834)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10831..10853)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10842..10858
                     /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(10866..10872,10873..10890,10891..10896))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10911..10932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11049..11066)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11220..11808)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11300..11319)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11979..12190,1..580,581..649))
                     /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"
ORIGIN
        1 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
       61 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
      121 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
      181 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
      241 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
      301 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
      361 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
      421 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
      481 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
      541 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
      601 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
      661 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
      721 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
      781 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
      841 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
      901 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
      961 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     1021 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     1081 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     1141 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     1201 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     1261 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     1321 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     1381 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg ggctaaccct
     1441 agaaagatag tctgcgtaaa attgacgcat gcattcttga aatattgctc tctctttcta
     1501 aatagcgcga atccgtcgct gtgcatttag gacatctcag tcgccgcttg gagctcccgt
     1561 gaggcgtgct tgtcaatgcg gtaagtgtca ctgattttga actataacga ccgcgtgagt
     1621 caaaatgacg catgattatc ttttacgtga cttttaagat ttaactcata cgataattat
     1681 attgttattt catgttctac ttacgtgata acttattata tatatatttt cttgttatag
     1741 atatcaacta gaatacgcgt aaggatctgc gatcgctccg gtgcccgtca gtgggcagag
     1801 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc
     1861 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     1921 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     1981 aacgggtttg ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc
     2041 gcccgccgcc ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc
     2101 gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga
     2161 ccgggccttt gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt
     2221 tgcctgaccc tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac
     2281 agatccaagc tgtgaccggc gcctactcta gagctagcga attcgccgcc accatgccta
     2341 agaaaaagag gaaggtgagc ggccgcagcc ttgccctgtc acttacagcc gaccagatgg
     2401 tttccgcgct tctcgacgct gaacctccaa ttctctattc cgaatacgac ccaaccaggc
     2461 cgttctccga ggcatctatg atgggtctgc tgacaaatct ggcagacagg gaactggtgc
     2521 acatgatcaa ttgggcgaag cgcgtacccg gattcgtcga tcttgcactc catgatcagg
     2581 tgcacttgct ggagtgcgct tggatggaga tcctcatgat cgggctggtg tggcggagta
     2641 tggaacaccc cggcaagttg ctgtttgcgc ctaacctcct gttggacagg aaccagggga
     2701 aatgtgtgga gggcggtgtg gaaatctttg acatgctcct cgctacctca agccggttta
     2761 ggatgatgaa tctgcagggc gaagagttcg tgtgtctcaa atctatcata ctgttgaaca
     2821 gcggagtcta caccttcctc tccagtactc tgaaatctct ggaggagaaa gatcatatcc
     2881 atcgcgtgct ggacaagata accgacacgt tgattcactt gatggccaaa gctgggctca
     2941 ctctgcaaca acaacatcag cgactggcac agctgttgct gattttgagc cacattcggc
     3001 acatgtccag caagagaatg gagcacctct atagtatgaa gtgcaagaac gtcgtacccc
     3061 tgtcagatct gcttcttgaa atgcttgatg cccaccggct gggatccggc ggatctggag
     3121 gtagtggtgg atctggaggt tctggtggct ccgacaagaa gtacagcatc ggcctggcca
     3181 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     3241 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggcgccc
     3301 tgctgttcga cagcggagaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     3361 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     3421 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     3481 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     3541 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     3601 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     3661 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     3721 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     3781 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     3841 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     3901 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     3961 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     4021 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     4081 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     4141 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     4201 tcttcgacca gagcaagaac ggctacgccg gctacatcga tggcggagcc agccaggaag
     4261 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     4321 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     4381 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     4441 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     4501 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     4561 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc gcccagagct
     4621 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     4681 acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg aaatacgtga
     4741 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaagcc atcgtggacc
     4801 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     4861 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     4921 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     4981 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     5041 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     5101 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     5161 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     5221 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     5281 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     5341 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     5401 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     5461 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     5521 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     5581 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     5641 accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg aaggacgact
     5701 ccatcgataa caaagtgctg actcggagcg acaagaaccg gggcaagagc gacaacgtgc
     5761 cctccgaaga ggtcgtgaag aagatgaaga actactggcg ccagctgctg aatgccaagc
     5821 tgattaccca gaggaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     5881 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     5941 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaacgac aaactgatcc
     6001 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     6061 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     6121 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     6181 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     6241 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     6301 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaacaggcg
     6361 agatcgtgtg ggataagggc cgggactttg ccaccgtgcg gaaagtgctg tctatgcccc
     6421 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     6481 tgcccaagag gaacagcgac aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     6541 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     6601 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     6661 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     6721 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     6781 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     6841 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     6901 ataatgagca gaaacagctg tttgtggaac agcacaaaca ctacctggac gagatcatcg
     6961 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaggtgc
     7021 tgagcgccta caacaagcac agagacaagc ctatcagaga gcaggccgag aatatcatcc
     7081 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     7141 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     7201 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gaccccaaga
     7261 agaagcgcaa ggtgcaccac caccaccacc acggaggagg tggaagcgga ggaggaggaa
     7321 gcggaggagg aggtagctgg cgcgccgtgt ctaagggcga agagctgatt aaggagaaca
     7381 tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag tgcacatccg
     7441 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     7501 gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc agcagaacct
     7561 tcatcaacca cacccagggc atccccgact tctttaagca gtccttccct gagggcttca
     7621 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     7681 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcccatcca
     7741 acggccctgt gatgcagaag aaaacactcg gctgggaggc caacaccgag atgctgtacc
     7801 ccgctgacgg cggcctggaa ggcagaaccg acatggccct gaagctcgtg ggcgggggcc
     7861 acctgatctg caacttcaag accacataca gatccaagaa acccgctaag aacctcaaga
     7921 tgcccggcgt ctactatgtg gaccacagac tggaaagaat caaggaggcc gacaaagaga
     7981 cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg
     8041 ggcacaaact taatggcatg gacgagctgt acaagttaat taacggaagc ggagctacta
     8101 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct atgagcgagc
     8161 tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact
     8221 tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca
     8281 aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct
     8341 acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct
     8401 tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg
     8461 ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg
     8521 tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca
     8581 ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc
     8641 tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc aagaaacccg
     8701 ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg
     8761 aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc
     8821 tccctagcaa actggggcac aagcttaatt agacgcgtat taacgactgt gccttctagt
     8881 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     8941 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     9001 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     9061 aggcatgctg gggatgcggt gggctctatg gcctctggcc tcgcacacat tccacatcca
     9121 ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc
     9181 ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg
     9241 aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg
     9301 taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc
     9361 tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc
     9421 gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt
     9481 tctcctcttc ctcatctccg ggcctttcga cctgcaatgg ccaagccttt gtctcaagaa
     9541 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     9601 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     9661 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     9721 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     9781 ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg
     9841 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     9901 gtgtgggagg gctaaaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     9961 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
    10021 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
    10081 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
    10141 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
    10201 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
    10261 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
    10321 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
    10381 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
    10441 cctcttccgc gtctccgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
    10501 ccgccggtac ctaaaagttt tgttacttta tagaagaaat tttgagtttt tgtttttttt
    10561 taataaataa ataaacataa ataaattgtt tgttgaattt attattagta tgtaagtgta
    10621 aatataataa aacttaatat ctattcaaat taataaataa acctcgatat acagaccgat
    10681 aaaacacatg cgtcaatttt acgcatgatt atctttaacg tacgtcacaa tatgattatc
    10741 tttctagggt taatctagta ttaatccagc ttttgttccc tttagtgagg gttaattgcg
    10801 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
    10861 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
    10921 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
    10981 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
    11041 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
    11101 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
    11161 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
    11221 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
    11281 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
    11341 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
    11401 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
    11461 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
    11521 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
    11581 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
    11641 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
    11701 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
    11761 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
    11821 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
    11881 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
    11941 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
    12001 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
    12061 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
    12121 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
    12181 cgcagaagtg
//
