LOCUS       sequence-375730-       15344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ef1a driven PE2 plasmid with pegRNA for editing C201R mutation in 
            KCNQ2 gene. See Addgene plasmid #184445.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15344)
  TITLE     All in one plasmids SCN1A and KCNQ2 mutations
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 15344)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAIO-Ef1a-PE2-GFP:KCNQ2-C201R
FEATURES             Location/Qualifiers
     source          1..15344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     169..190
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             245..265
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             274..291
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    329..917
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(382..402)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(800..811)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1124..1757
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(1885..1901)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1885..1901)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1898..1920)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1909..1925
                     /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(1933..1939,1940..1957,1958..1963))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1978..1999
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2116..2133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2287..2875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2367..2386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3046..3837,3838..3906))
                     /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     3669..3688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3907..4011)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4059..4193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4096..4115)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4150..4169
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             4361..4994
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5041..5166
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5663..5896
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6081..6125
                     /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"
     misc_feature    6388..6503
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        6570..6818
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6570..6590
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6741..6760
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6839..6914
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        7165..8347
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          7396..8338
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     8295..8315
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     8411..8430
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8411..8429
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             8479..8499
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(8500..11013,11014..11016,11017..12600)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8500..12600
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             14773..14793
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             14803..14859
                     /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             join(14860..14862,14863..14865,14866..15344,1..232)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(14860..14862,14863..14865,14866..15344,1..232)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(15166..15185)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
       61 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
      121 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
      181 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtctggtgg
      241 ttctcccaag aagaagagga aagtctaacc ggtcatcatc accatcacca ttgagtttta
      301 ccaccggtgg aattcacgcg tctggaacaa tcaacctctg gattacaaaa tttgtgaaag
      361 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
      421 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
      481 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
      541 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
      601 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
      661 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
      721 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
      781 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
      841 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
      901 ttgggccgcc tccccgcctg gaattaattc tgcagtcgag acctagaaaa acatggagca
      961 atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag
     1021 gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag
     1081 gcagctgtag atcttagcca ctttttaaaa gaaaagaggg gactggaagg gctaattcac
     1141 tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct
     1201 gattagcaga actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc
     1261 tacaagctag taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc
     1321 agcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtgttagag
     1381 tggaggtttg acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac
     1441 ttcaagaact gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg
     1501 aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct
     1561 gctttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     1621 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     1681 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     1741 gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc
     1801 aaagaaatga atatcagaga gtgagaggcc ttgacattgc tagcgtttac cgtcgacctc
     1861 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1921 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1981 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2041 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2101 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2161 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2221 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2281 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2341 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2401 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2461 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2521 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2581 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2641 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2701 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2761 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2821 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2881 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2941 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3001 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3061 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     3961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4021 ccgaaaagtg ccacctgacg tcgacggatc gggagatcaa cttgtttatt gcagcttata
     4081 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4141 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
     4201 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
     4261 attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa
     4321 gaaattgtat ttgttaaata tgtactacaa acttagtagt tggaagggct aattcactcc
     4381 caaagaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     4441 tagcagaact acacaccagg gccaggggtc agatatccac tgacctttgg atggtgctac
     4501 aagctagtac cagttgagcc agataaggta gaagaggcca ataaaggaga gaacaccagc
     4561 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt gttagagtgg
     4621 aggtttgaca gccgcctagc atttcatcac gtggcccgag agctgcatcc ggagtacttc
     4681 aagaactgct gatatcgagc ttgctacaag ggactttccg ctggggactt tccagggagg
     4741 cgtggcctgg gcgggactgg ggagtggcga gccctcagat cctgcatata agcagctgct
     4801 ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     4861 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     4921 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     4981 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
     5041 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
     5101 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
     5161 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     5221 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     5281 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     5341 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     5401 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     5461 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgctgatct
     5521 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     5581 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     5641 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     5701 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     5761 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     5821 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     5881 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     5941 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     6001 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     6061 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     6121 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     6181 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     6241 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg
     6301 acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat
     6361 tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag gggggattgg ggggtacagt
     6421 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     6481 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttat
     6541 cgatggtccg atttcagcag gtgccatgtg agggcctatt tcccatgatt ccttcatatt
     6601 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     6661 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     6721 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     6781 tcttggcttt atatatcttg tggaaaggac gaaacaccgc agaatctgca ggaagcacgt
     6841 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     6901 caccgagtcg gtgctccgga gcctgcgctt cctgcagatt ttttttagag ccacctgcgg
     6961 gcagatcgaa cctgcaggtt ccactgggac gattacatgc atctggatcg atgagtaatt
     7021 catacaaaag gactcgcccc tgccttgggg aatcccaggg accgtcgtta aactcccact
     7081 aacgtagaac ccagagatcg ctgcgttccc gccccctcac ccgcccgctc tcgtcatcac
     7141 tgaggtggag aagagcatgc gtgaggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     7201 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
     7261 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     7321 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     7381 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
     7441 tatggccctt gcgtgccttg aattacttcc acgcccctgg ctgcagtacg tgattcttga
     7501 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
     7561 ttcgcctcgt gcttgagttg aggcctggct tgggcgctgg ggccgccgcg tgcgaatctg
     7621 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
     7681 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
     7741 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     7801 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     7861 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     7921 ggcggcaagg ctggcccggt cggcgccagt tgcgtgagcg gaaagatggc cgcttcccgg
     7981 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     8041 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     8101 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt
     8161 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     8221 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     8281 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     8341 gtcgtgagga tctatttccg gtgaattggt ttaaacgcga tcgcctcgag ggatcctcta
     8401 gagcggccgc taatacgact cactataggg agagccgcca ccatgaaacg gacagccgac
     8461 ggaagcgagt tcgagtcacc aaagaagaag cggaaagtcg acaagaagta cagcatcggc
     8521 ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     8581 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     8641 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     8701 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     8761 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     8821 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     8881 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     8941 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     9001 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     9061 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     9121 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     9181 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     9241 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     9301 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     9361 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     9421 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     9481 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     9541 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     9601 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     9661 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     9721 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     9781 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     9841 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     9901 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     9961 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
    10021 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
    10081 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
    10141 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
    10201 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
    10261 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
    10321 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
    10381 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
    10441 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
    10501 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
    10561 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
    10621 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
    10681 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
    10741 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
    10801 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
    10861 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
    10921 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
    10981 gacatcaacc ggctgtccga ctacgatgtg gacgctatcg tgcctcagag ctttctgaag
    11041 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
    11101 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
    11161 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
    11221 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
    11281 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
    11341 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
    11401 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
    11461 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
    11521 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
    11581 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
    11641 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
    11701 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
    11761 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
    11821 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
    11881 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
    11941 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
    12001 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
    12061 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
    12121 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
    12181 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
    12241 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
    12301 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
    12361 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
    12421 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
    12481 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
    12541 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac
    12601 tctggaggat ctagcggagg atcctctggc agcgagacac caggaacaag cgagtcagca
    12661 acaccagaga gcagtggcgg cagcagcggc ggcagcagca ccctaaatat agaagatgag
    12721 tatcggctac atgagacctc aaaagagcca gatgtttctc tagggtccac atggctgtct
    12781 gattttcctc aggcctgggc ggaaaccggg ggcatgggac tggcagttcg ccaagctcct
    12841 ctgatcatac ctctgaaagc aacctctacc cccgtgtcca taaaacaata ccccatgtca
    12901 caagaagcca gactggggat caagccccac atacagagac tgttggacca gggaatactg
    12961 gtaccctgcc agtccccctg gaacacgccc ctgctacccg ttaagaaacc agggactaat
    13021 gattataggc ctgtccagga tctgagagaa gtcaacaagc gggtggaaga catccacccc
    13081 accgtgccca acccttacaa cctcttgagc gggctcccac cgtcccacca gtggtacact
    13141 gtgcttgatt taaaggatgc ctttttctgc ctgagactcc accccaccag tcagcctctc
    13201 ttcgcctttg agtggagaga tccagagatg ggaatctcag gacaattgac ctggaccaga
    13261 ctcccacagg gtttcaaaaa cagtcccacc ctgtttaatg aggcactgca cagagaccta
    13321 gcagacttcc ggatccagca cccagacttg atcctgctac agtacgtgga tgacttactg
    13381 ctggccgcca cttctgagct agactgccaa caaggtactc gggccctgtt acaaacccta
    13441 gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa tttgccagaa acaggtcaag
    13501 tatctggggt atcttctaaa agagggtcag agatggctga ctgaggccag aaaagagact
    13561 gtgatggggc agcctactcc gaagacccct cgacaactaa gggagttcct agggaaggca
    13621 ggcttctgtc gcctcttcat ccctgggttt gcagaaatgg cagcccccct gtaccctctc
    13681 accaaaccgg ggactctgtt taattggggc ccagaccaac aaaaggccta tcaagaaatc
    13741 aagcaagctc ttctaactgc cccagccctg gggttgccag atttgactaa gccctttgaa
    13801 ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc taacgcaaaa actgggacct
    13861 tggcgtcggc cggtggccta cctgtccaaa aagctagacc cagtagcagc tgggtggccc
    13921 ccttgcctac ggatggtagc agccattgcc gtactgacaa aggatgcagg caagctaacc
    13981 atgggacagc cactagtcat tctggccccc catgcagtag aggcactagt caaacaaccc
    14041 cccgaccgct ggctttccaa cgcccggatg actcactatc aggccttgct tttggacacg
    14101 gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg ctacgctgct cccactgcct
    14161 gaggaagggc tgcaacacaa ctgccttgat atcctggccg aagcccacgg aacccgaccc
    14221 gacctaacgg accagccgct cccagacgcc gaccacacct ggtacacgga tggaagcagt
    14281 ctcttacaag agggacagcg taaggcggga gctgcggtga ccaccgagac cgaggtaatc
    14341 tgggctaaag ccctgccagc cgggacatcc gctcagcggg ctgaactgat agcactcacc
    14401 caggccctaa agatggcaga aggtaagaag ctaaatgttt atactgatag ccgttatgct
    14461 tttgctactg cccatatcca tggagaaata tacagaaggc gtgggtggct cacatcagaa
    14521 ggcaaagaga tcaaaaataa agacgagatc ttggccctac taaaagccct ctttctgccc
    14581 aaaagactta gcataatcca ttgtccagga catcaaaagg gacacagcgc cgaggctaga
    14641 ggcaaccgga tggctgacca agcggcccga aaggcagcca tcacagagac tccagacacc
    14701 tctaccctcc tcatagaaaa ttcatcaccc tctggcggct caaaaagaac cgccgacggc
    14761 agcgaattcg agcccaagaa gaagaggaaa gtcggaagcg gagctactaa cttcagcctg
    14821 ctgaagcagg ctggagacgt ggaggagaac cctggaccta tggtgagcaa gggcgaggag
    14881 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
    14941 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
    15001 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctat
    15061 ggagtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
    15121 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
    15181 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
    15241 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
    15301 agccacaacg tctatatcat ggccgacaag cagaagaacg gcat
//
