LOCUS       sequence-376807-       15344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ef1a driven PE2 with pegRNA for editing C201R mutation (including 
            silent PAM mutation) in KCNQ2. 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  JOURNAL   Unpublished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            "Unpublished"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_pp
FEATURES             Location/Qualifiers
     source          1..15344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             149..169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             179..235
                     /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(236..238,239..241,242..952)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(236..238,239..241,242..952)
                     /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(542..561)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     889..910
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             965..985
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             994..1011
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    1049..1637
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1102..1122)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1520..1531)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1844..2477
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(2605..2621)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2605..2621)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2618..2640)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2629..2645
                     /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(2653..2659,2660..2677,2678..2683))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2698..2719
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2836..2853)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3007..3595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3087..3106)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3766..4557,4558..4626))
                     /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     4389..4408
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4627..4731)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4779..4913
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4816..4835)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4870..4889
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5081..5714
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5761..5886
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6383..6616
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6801..6845
                     /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    7108..7223
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        7290..7538
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7290..7310
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7461..7480
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7559..7634
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        7885..9067
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          8116..9058
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     9015..9035
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     9131..9150
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9131..9149
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             9199..9219
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(9220..11733,11734..11736,11737..13320)
                     /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             9220..13320
                     /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"
ORIGIN
        1 cagcgccgag gctagaggca accggatggc tgaccaagcg gcccgaaagg cagccatcac
       61 agagactcca gacacctcta ccctcctcat agaaaattca tcaccctctg gcggctcaaa
      121 aagaaccgcc gacggcagcg aattcgagcc caagaagaag aggaaagtcg gaagcggagc
      181 tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg gacctatggt
      241 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      301 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      361 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      421 gaccaccctg acctatggag tgcagtgctt cagccgctac cccgaccaca tgaagcagca
      481 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      541 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
      601 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
      661 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
      721 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
      781 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
      841 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
      901 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtctggtgg
      961 ttctcccaag aagaagagga aagtctaacc ggtcatcatc accatcacca ttgagtttta
     1021 ccaccggtgg aattcacgcg tctggaacaa tcaacctctg gattacaaaa tttgtgaaag
     1081 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     1141 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     1201 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     1261 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     1321 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     1381 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     1441 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     1501 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     1561 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     1621 ttgggccgcc tccccgcctg gaattaattc tgcagtcgag acctagaaaa acatggagca
     1681 atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag
     1741 gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag
     1801 gcagctgtag atcttagcca ctttttaaaa gaaaagaggg gactggaagg gctaattcac
     1861 tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct
     1921 gattagcaga actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc
     1981 tacaagctag taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc
     2041 agcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtgttagag
     2101 tggaggtttg acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac
     2161 ttcaagaact gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg
     2221 aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct
     2281 gctttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     2341 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     2401 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     2461 gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc
     2521 aaagaaatga atatcagaga gtgagaggcc ttgacattgc tagcgtttac cgtcgacctc
     2581 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     2641 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     2701 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2761 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2821 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2881 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2941 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3001 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3061 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3121 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3181 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3241 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3301 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3361 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3421 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3481 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3541 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3601 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3661 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3721 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3781 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3841 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3901 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3961 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4021 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4081 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4141 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4201 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4261 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4321 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4381 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4441 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4501 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4561 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4621 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4681 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4741 ccgaaaagtg ccacctgacg tcgacggatc gggagatcaa cttgtttatt gcagcttata
     4801 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4861 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
     4921 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
     4981 attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa
     5041 gaaattgtat ttgttaaata tgtactacaa acttagtagt tggaagggct aattcactcc
     5101 caaagaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     5161 tagcagaact acacaccagg gccaggggtc agatatccac tgacctttgg atggtgctac
     5221 aagctagtac cagttgagcc agataaggta gaagaggcca ataaaggaga gaacaccagc
     5281 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt gttagagtgg
     5341 aggtttgaca gccgcctagc atttcatcac gtggcccgag agctgcatcc ggagtacttc
     5401 aagaactgct gatatcgagc ttgctacaag ggactttccg ctggggactt tccagggagg
     5461 cgtggcctgg gcgggactgg ggagtggcga gccctcagat cctgcatata agcagctgct
     5521 ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     5581 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     5641 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     5701 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
     5761 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
     5821 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
     5881 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     5941 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     6001 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     6061 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     6121 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     6181 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgctgatct
     6241 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     6301 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     6361 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     6421 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     6481 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     6541 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     6601 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     6661 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     6721 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     6781 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     6841 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     6901 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     6961 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg
     7021 acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat
     7081 tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag gggggattgg ggggtacagt
     7141 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     7201 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttat
     7261 cgatggtccg atttcagcag gtgccatgtg agggcctatt tcccatgatt ccttcatatt
     7321 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     7381 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     7441 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     7501 tcttggcttt atatatcttg tggaaaggac gaaacaccgc agaatctgca ggaagcacgt
     7561 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     7621 caccgagtcg gtgctccgga gtctgcgctt cctgcagatt ttttttagag ccacctgcgg
     7681 gcagatcgaa cctgcaggtt ccactgggac gattacatgc atctggatcg atgagtaatt
     7741 catacaaaag gactcgcccc tgccttgggg aatcccaggg accgtcgtta aactcccact
     7801 aacgtagaac ccagagatcg ctgcgttccc gccccctcac ccgcccgctc tcgtcatcac
     7861 tgaggtggag aagagcatgc gtgaggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     7921 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
     7981 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     8041 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     8101 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
     8161 tatggccctt gcgtgccttg aattacttcc acgcccctgg ctgcagtacg tgattcttga
     8221 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
     8281 ttcgcctcgt gcttgagttg aggcctggct tgggcgctgg ggccgccgcg tgcgaatctg
     8341 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
     8401 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
     8461 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     8521 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     8581 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     8641 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
     8701 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     8761 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     8821 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt
     8881 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     8941 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     9001 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     9061 gtcgtgagga tctatttccg gtgaattggt ttaaacgcga tcgcctcgag ggatcctcta
     9121 gagcggccgc taatacgact cactataggg agagccgcca ccatgaaacg gacagccgac
     9181 ggaagcgagt tcgagtcacc aaagaagaag cggaaagtcg acaagaagta cagcatcggc
     9241 ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     9301 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     9361 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     9421 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     9481 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     9541 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     9601 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     9661 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     9721 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     9781 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     9841 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     9901 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     9961 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
    10021 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
    10081 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
    10141 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
    10201 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
    10261 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
    10321 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
    10381 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
    10441 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
    10501 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
    10561 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
    10621 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
    10681 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
    10741 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
    10801 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
    10861 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
    10921 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
    10981 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
    11041 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
    11101 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
    11161 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
    11221 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
    11281 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
    11341 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
    11401 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
    11461 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
    11521 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
    11581 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
    11641 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
    11701 gacatcaacc ggctgtccga ctacgatgtg gacgctatcg tgcctcagag ctttctgaag
    11761 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
    11821 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
    11881 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
    11941 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
    12001 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
    12061 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
    12121 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
    12181 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
    12241 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
    12301 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
    12361 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
    12421 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
    12481 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
    12541 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
    12601 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
    12661 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
    12721 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
    12781 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
    12841 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
    12901 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
    12961 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
    13021 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
    13081 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
    13141 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
    13201 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
    13261 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac
    13321 tctggaggat ctagcggagg atcctctggc agcgagacac caggaacaag cgagtcagca
    13381 acaccagaga gcagtggcgg cagcagcggc ggcagcagca ccctaaatat agaagatgag
    13441 tatcggctac atgagacctc aaaagagcca gatgtttctc tagggtccac atggctgtct
    13501 gattttcctc aggcctgggc ggaaaccggg ggcatgggac tggcagttcg ccaagctcct
    13561 ctgatcatac ctctgaaagc aacctctacc cccgtgtcca taaaacaata ccccatgtca
    13621 caagaagcca gactggggat caagccccac atacagagac tgttggacca gggaatactg
    13681 gtaccctgcc agtccccctg gaacacgccc ctgctacccg ttaagaaacc agggactaat
    13741 gattataggc ctgtccagga tctgagagaa gtcaacaagc gggtggaaga catccacccc
    13801 accgtgccca acccttacaa cctcttgagc gggctcccac cgtcccacca gtggtacact
    13861 gtgcttgatt taaaggatgc ctttttctgc ctgagactcc accccaccag tcagcctctc
    13921 ttcgcctttg agtggagaga tccagagatg ggaatctcag gacaattgac ctggaccaga
    13981 ctcccacagg gtttcaaaaa cagtcccacc ctgtttaatg aggcactgca cagagaccta
    14041 gcagacttcc ggatccagca cccagacttg atcctgctac agtacgtgga tgacttactg
    14101 ctggccgcca cttctgagct agactgccaa caaggtactc gggccctgtt acaaacccta
    14161 gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa tttgccagaa acaggtcaag
    14221 tatctggggt atcttctaaa agagggtcag agatggctga ctgaggccag aaaagagact
    14281 gtgatggggc agcctactcc gaagacccct cgacaactaa gggagttcct agggaaggca
    14341 ggcttctgtc gcctcttcat ccctgggttt gcagaaatgg cagcccccct gtaccctctc
    14401 accaaaccgg ggactctgtt taattggggc ccagaccaac aaaaggccta tcaagaaatc
    14461 aagcaagctc ttctaactgc cccagccctg gggttgccag atttgactaa gccctttgaa
    14521 ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc taacgcaaaa actgggacct
    14581 tggcgtcggc cggtggccta cctgtccaaa aagctagacc cagtagcagc tgggtggccc
    14641 ccttgcctac ggatggtagc agccattgcc gtactgacaa aggatgcagg caagctaacc
    14701 atgggacagc cactagtcat tctggccccc catgcagtag aggcactagt caaacaaccc
    14761 cccgaccgct ggctttccaa cgcccggatg actcactatc aggccttgct tttggacacg
    14821 gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg ctacgctgct cccactgcct
    14881 gaggaagggc tgcaacacaa ctgccttgat atcctggccg aagcccacgg aacccgaccc
    14941 gacctaacgg accagccgct cccagacgcc gaccacacct ggtacacgga tggaagcagt
    15001 ctcttacaag agggacagcg taaggcggga gctgcggtga ccaccgagac cgaggtaatc
    15061 tgggctaaag ccctgccagc cgggacatcc gctcagcggg ctgaactgat agcactcacc
    15121 caggccctaa agatggcaga aggtaagaag ctaaatgttt atactgatag ccgttatgct
    15181 tttgctactg cccatatcca tggagaaata tacagaaggc gtgggtggct cacatcagaa
    15241 ggcaaagaga tcaaaaataa agacgagatc ttggccctac taaaagccct ctttctgccc
    15301 aaaagactta gcataatcca ttgtccagga catcaaaagg gaca
//
