LOCUS       sequence-466778-       14033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EF1-alpha driven co-expression of the consitutively active kinase 
            activity recorder positive control Kinprola_on 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 14033)
  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 14033)
  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_on-mEGFP_bGH-PA
            term_...
FEATURES             Location/Qualifiers
     source          1..14033
                     /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(2756..2758,2759..2761,2762..3475)
                     /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(3062..3081)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3409..3430
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3507..3524)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3513..3737
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3892..4125
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4310..4354
                     /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     4532..4552
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    4753..4870
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4939..5150
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5170..5179
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5179..5202
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5209..5229
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5254..9354
                     /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             5254..9354
                     /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             9355..9402
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9418..9471
                     /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             9472..9870
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9886..10474
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9939..9959)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10357..10368)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10546..10779
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10857..10978
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10894..10913)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10948..10967
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      11018..11153
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(11173..11192)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11174..11192)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11202..11219)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11202..11218)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11211..11233)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11360..11815
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11447..11466)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11657..11678
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11841..11945
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11946..12014,12015..12806)
                     /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(12164..12183)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12977..13565
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13466..13485
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13719..13736
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13853..13874
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13889..13894,13895..13912,13913..13919)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13927..13943
                     /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     13932..13954
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13951..13967
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13951..13967
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13986..14006
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13988..14006
                     /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 cttttgcccg cgagaccttc caggccttcc gcaccaccga
     1261 cgtcggccgc aagctgatca tcgatcagaa cgtttttatc gagggtacgc tgccgatggg
     1321 tgtcgtccgc ccgctgactg aagtcgagat ggaccattac cgcgagccgt tcctgaatcc
     1381 tgttgaccgc gagccactgt ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc
     1441 gaacatcgtc gcgctggtcg aagaatacat ggactggctg caccagtccc ctgtcccgaa
     1501 gctgctgttc tggggcaccc caggcgttct gatcccaccg gccgaagccg ctcgcctggc
     1561 caaaagcctg cctaactgca aggctgtgga catcggcccg ggtctgaatc tgctgcaaga
     1621 agacaacccg gacctgatcg gcagcgagat cgcgcgctgg ctgtctactc tggagattgg
     1681 tggcaccggt ggctctggcg gtaccggtgg ctctggcggt tctatcggta ctggctttcc
     1741 attcgacccc cattatgtgg aagtcctggg cgagcgcatg cactacgtcg atgttggtcc
     1801 gcgcgatggc acccctgtgc tgttcctgca cggtaacccg acctcctcct acgtgtggcg
     1861 caacatcatc ccgcatgttg caccgaccca tcgctgcatt gctccagacc tgatcggtat
     1921 gggcaaatcc gacaaaccag acctgggtta tttcttcgac gaccacgtcc gcttcatgga
     1981 tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg actggggctc
     2041 cgctctgggt ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat
     2101 ggagttcatc cgccctatcc cgacctggga cgaatgggct ccgggttctc tgcgtcgcgc
     2161 caccctggtg gaccctcctc ctccgccacc gccaccaccg ccgccgccgc caccaccacc
     2221 tcctccgcct cctccaccac caccgccacc gcctccacca cctggcggca gcgcctgtga
     2281 ctatcattta ggaaacattt caagactgtc aaataagcat ttccaaatac tactaggaga
     2341 agacggtaac cttttattga atgacatttc cactaatggg acctggttaa atgggcaaaa
     2401 agtcgagaag aacagctatc agttactgtc tcaaggtgat gaaataaccg ttggtgtagg
     2461 cgtggaatca gatattttat ctctggtcat tttcataaac gacaaattta agcagtgcct
     2521 cgagcagaac aaagttgatc gcggtggcgg tatgcataag ttttctcaag aacagatcgg
     2581 cgaaaacatt gtgtgcaggg tcatttgtac cacgggtcaa attcccatcc gagatttgtc
     2641 agctgatatt tcacaagtgc ttaaggaaaa acgatccata aagaaagttt ggacatttgg
     2701 tagaaaccca ggttctgcca aggagacctt ccaggccttc cgcagcggag gttctatggt
     2761 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2821 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2881 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2941 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     3001 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3061 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3121 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     3181 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     3241 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     3301 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3361 gagcacccag tccaaactga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3421 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaatctag
     3481 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3541 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3601 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3661 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3721 tgcggtgggc tctatggggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     3781 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     3841 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     3901 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     3961 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     4021 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     4081 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     4141 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     4201 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     4261 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     4321 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     4381 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     4441 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     4501 gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata
     4561 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     4621 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     4681 tttaaaatta tgttttaaaa tggactatca taaattctag atcttgagac aaatggcagt
     4741 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     4801 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     4861 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgagtg
     4921 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     4981 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     5041 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     5101 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac
     5161 gcgggatccg ccaccatgga ttacaaagac gatgacgata agatggcccc aaagaagaag
     5221 cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac
     5281 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     5341 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     5401 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     5461 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     5521 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     5581 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     5641 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     5701 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     5761 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     5821 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     5881 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     5941 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     6001 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     6061 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     6121 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     6181 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     6241 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     6301 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     6361 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     6421 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     6481 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     6541 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     6601 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     6661 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     6721 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     6781 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     6841 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     6901 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     6961 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     7021 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     7081 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     7141 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     7201 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     7261 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     7321 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     7381 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     7441 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     7501 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     7561 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     7621 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     7681 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     7741 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac
     7801 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     7861 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     7921 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     7981 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     8041 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     8101 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     8161 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     8221 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     8281 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     8341 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     8401 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     8461 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     8521 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     8581 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     8641 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     8701 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     8761 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     8821 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     8881 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     8941 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     9001 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     9061 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     9121 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     9181 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     9241 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
     9301 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaagcgt
     9361 cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga aagctagcgg cagcggcgag
     9421 ggcagaggaa gtctgctaac atgcggtgat gtcgaagaaa atccaggccc aatggccaag
     9481 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     9541 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     9601 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     9661 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     9721 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     9781 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     9841 ctgccctctg gttatgtgtg ggagggctaa acgcgttaag tcgacaatca acctctggat
     9901 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     9961 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
    10021 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
    10081 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
    10141 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
    10201 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
    10261 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
    10321 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
    10381 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
    10441 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
    10501 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
    10561 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
    10621 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
    10681 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
    10741 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt agttcatgtc
    10801 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
    10861 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
    10921 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
    10981 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
    11041 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
    11101 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
    11161 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
    11221 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
    11281 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
    11341 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
    11401 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
    11461 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
    11521 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
    11581 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
    11641 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
    11701 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
    11761 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
    11821 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
    11881 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
    11941 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
    12001 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
    12061 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
    12121 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
    12181 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
    12241 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
    12301 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
    12361 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
    12421 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
    12481 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
    12541 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
    12601 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
    12661 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
    12721 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
    12781 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
    12841 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
    12901 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
    12961 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
    13021 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
    13081 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
    13141 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
    13201 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
    13261 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
    13321 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
    13381 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
    13441 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
    13501 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
    13561 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
    13621 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
    13681 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
    13741 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
    13801 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
    13861 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
    13921 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
    13981 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taa
//
