LOCUS       sequence-466777-       13997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EF1-alpha driven co-expression of the inactive PKA activity recorder
            negative control with T/A mutation Kinprola_PKA_T/A fused to mEGFP 
            and Cas9 expression through lentivirus transduction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13997)
  AUTHORS   Sun DE, Ng SW, Zheng Y, Xie S, Schwan N, Breuer P, Hoffmann DC, 
            Michel J, Azorin DD, Boonekamp KE, Winkler F, Wick W, Boutros M, Li 
            Y, Johnsson K
  TITLE     Molecular recording of cellular protein kinase activity with 
            chemical labeling.
  JOURNAL   Nat Chem Biol. 2025 Jul 10. doi: 10.1038/s41589-025-01949-6.
  PUBMED    40640555
REFERENCE   2  (bases 1 to 13997)
  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 Chem 
            Biol. 2025 Jul 10. doi: 10.1038/s41589-025-01949-6."
COMMENT     Sequence Label: pLenti_EF1-alpha-NES-Kinprola_PKA_T/A-mEGFP_bGH-PA
            ...
FEATURES             Location/Qualifiers
     source          1..13997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     promoter        923..1134
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             join(2720..2722,2723..2725,2726..3439)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3026..3045)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3373..3394
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3471..3488)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3477..3701
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3856..4089
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4274..4318
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     primer_bind     4496..4516
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    4717..4834
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4903..5114
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5134..5143
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5143..5166
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5173..5193
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5218..9318
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5218..9318
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             9319..9366
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9382..9435
                     /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             9436..9834
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9850..10438
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9903..9923)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10321..10332)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10510..10743
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10821..10942
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10858..10877)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10912..10931
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10982..11117
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(11137..11156)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11138..11156)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11166..11183)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11166..11182)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11175..11197)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11324..11779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11411..11430)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11621..11642
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11805..11909
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11910..11978,11979..12770)
                     /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     complement(12128..12147)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12941..13529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13430..13449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13683..13700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13817..13838
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13853..13858,13859..13876,13877..13883)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13891..13907
                     /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)."
     primer_bind     13896..13918
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13915..13931
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13915..13931
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13950..13970
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13952..13970
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgggcagag cgcacatcgc ccacagtccc cgagaagttg
      961 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     1021 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     1081 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg
     1141 tgacgcggga tccgccacca tgctgcagaa cgagcttgct cttaagttgg ctggacttga
     1201 tattaacaag actggaggtt ctgacgtcgg ccgcaagctg atcatcgatc agaacgtttt
     1261 tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca
     1321 ttaccgcgag ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga
     1381 gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg
     1441 gctgcaccag tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc
     1501 accggccgaa gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg
     1561 cccgggtctg aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg
     1621 ctggctgtct actctggaga ttggtggcac cggtggctct ggcggtaccg gtggctctgg
     1681 cggttctatc ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg
     1741 catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa
     1801 cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg
     1861 cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt
     1921 cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt
     1981 cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga
     2041 gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg
     2101 ggctccgggt tctctgcgtc gcgccgccct ggtggaccct cctcctccgc caccgccacc
     2161 accgccgccg ccgccaccac cacctcctcc gcctcctcca ccaccaccgc caccgcctcc
     2221 accacctggc ggcagcgcct gtgactatca tttaggaaac atttcaagac tgtcaaataa
     2281 gcatttccaa atactactag gagaagacgg taacctttta ttgaatgaca tttccactaa
     2341 tgggacctgg ttaaatgggc aaaaagtcga gaagaacagc tatcagttac tgtctcaagg
     2401 tgatgaaata accgttggtg taggcgtgga atcagatatt ttatctctgg tcattttcat
     2461 aaacgacaaa tttaagcagt gcctcgagca gaacaaagtt gatcgcggtg gcggtatgca
     2521 taagttttct caagaacaga tcggcgaaaa cattgtgtgc agggtcattt gtaccacggg
     2581 tcaaattccc atccgagatt tgtcagctga tatttcacaa gtgcttaagg aaaaacgatc
     2641 cataaagaaa gtttggacat ttggtagaaa cccaggttct gccaaggaga ccttccaggc
     2701 cttccgcagc ggaggttcta tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     2761 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2821 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2881 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2941 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     3001 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     3061 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     3121 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     3181 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     3241 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     3301 gctgctgccc gacaaccact acctgagcac ccagtccaaa ctgagcaaag accccaacga
     3361 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3421 ggacgagctg tacaagtaat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3481 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3541 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3601 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3661 agacaatagc aggcatgctg gggatgcggt gggctctatg gggccgctga tcttcagacc
     3721 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     3781 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     3841 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     3901 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     3961 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     4021 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     4081 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     4141 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     4201 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     4261 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     4321 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     4381 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     4441 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg
     4501 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     4561 tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     4621 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcataaatt
     4681 ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg
     4741 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga
     4801 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga
     4861 tccactttgg cgccggctcg agtggctccg gtgcccgtca gtgggcagag cgcacatcgc
     4921 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
     4981 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     5041 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     5101 ccgccagaac acaggtgtcg tgacgcggga tccgccacca tggattacaa agacgatgac
     5161 gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac
     5221 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
     5281 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
     5341 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
     5401 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
     5461 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
     5521 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
     5581 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
     5641 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
     5701 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
     5761 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     5821 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     5881 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
     5941 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     6001 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     6061 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     6121 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     6181 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     6241 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     6301 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     6361 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     6421 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     6481 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     6541 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     6601 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     6661 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     6721 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     6781 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     6841 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     6901 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     6961 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     7021 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     7081 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     7141 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     7201 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     7261 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     7321 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     7381 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     7441 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     7501 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     7561 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     7621 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     7681 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     7741 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     7801 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     7861 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     7921 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     7981 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     8041 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     8101 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     8161 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     8221 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     8281 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     8341 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     8401 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     8461 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     8521 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     8581 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     8641 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     8701 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     8761 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     8821 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     8881 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     8941 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     9001 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     9061 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     9121 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     9181 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     9241 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     9301 tctcagctgg gaggcgacaa gcgtcctgct gctactaaga aagctggtca agctaagaaa
     9361 aagaaagcta gcggcagcgg cgagggcaga ggaagtctgc taacatgcgg tgatgtcgaa
     9421 gaaaatccag gcccaatggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     9481 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     9541 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     9601 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     9661 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     9721 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     9781 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaaacgcgt
     9841 taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     9901 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     9961 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
    10021 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
    10081 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
    10141 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
    10201 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
    10261 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
    10321 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
    10381 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt
    10441 cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
    10501 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
    10561 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
    10621 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
    10681 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
    10741 gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
    10801 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
    10861 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
    10921 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
    10981 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
    11041 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
    11101 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
    11161 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
    11221 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
    11281 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
    11341 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
    11401 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
    11461 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
    11521 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
    11581 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
    11641 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
    11701 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
    11761 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
    11821 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
    11881 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
    11941 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
    12001 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
    12061 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
    12121 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
    12181 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
    12241 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
    12301 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
    12361 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
    12421 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
    12481 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
    12541 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
    12601 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
    12661 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
    12721 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
    12781 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
    12841 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
    12901 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
    12961 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
    13021 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
    13081 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
    13141 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
    13201 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
    13261 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
    13321 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
    13381 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
    13441 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
    13501 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
    13561 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
    13621 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
    13681 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
    13741 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
    13801 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
    13861 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
    13921 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
    13981 tggagctgca agcttaa
//
