LOCUS       sequence-488452-       14092 bp    DNA     circular SYN 06-OCT-2025
DEFINITION  pLV-U6-[hSNCA gRNA 4]-EFSNC-dSpCas9-DNMT3a-P2A-PURO-WPRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14092)
  TITLE     SNCA-targeted epigenome therapy for Parkinson's disease alleviates 
            pathological and behavioral perturbations in a mouse model
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 14092)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pBK546
FEATURES             Location/Qualifiers
     source          1..14092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2634..2882
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2634..2654
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2805..2824
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2903..2978
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3040..3251
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3259..3268
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3268..3333
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3340..3360
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3385..7485
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYRDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7486..7533
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7561..8493
                     /codon_start=1
                     /product="catalytic domain of DNA methyltransferase 3A"
                     /label=DNMT3A catalytic domain
                     /translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG
                     LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI
                     GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVS
                     DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR
                     IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR
                     LARQRLLGRSWSVPVIRHLFAPLKEYFACV"
     CDS             8515..8571
                     /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             8572..9168
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     9065..9085
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9184..9772
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9237..9257)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9655..9666)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9844..10077
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10103..10120)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10109..10333
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10379..10807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10466..10485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10676..10697
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10816..10836)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10821..11150
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11001..11136
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11063..11082
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11198..11245
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11264..11638
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    11768..11889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11805..11824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11859..11878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11938..11954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11938..11954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11951..11973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11962..11978
                     /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(11986..11992,11993..12010,12011..12016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12031..12052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12169..12186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12340..12928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12420..12439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13099..13890,13891..13959))
                     /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     13722..13741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13960..14064)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta atgggcggga cgttaacggg gcggaacggt accgagggcc
     2641 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
     2701 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     2761 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     2821 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     2881 ccggagacgt gtacacgtct ctgttttaga gctagaaata gcaagttaaa ataaggctag
     2941 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgaattc gctagctagg
     3001 tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     3061 cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc
     3121 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     3181 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     3241 ccagaacaca ggaccggtgc caccatggac tataaggacc acgacggaga ctacaaggat
     3301 catgatattg attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc
     3361 ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctggc catcggcacc
     3421 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     3481 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     3541 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     3601 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     3661 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     3721 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     3781 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     3841 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     3901 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     3961 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     4021 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     4081 aagaatggcc tgttcggcaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     4141 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     4201 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     4261 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     4321 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     4381 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     4441 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     4501 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     4561 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     4621 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     4681 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     4741 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     4801 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     4861 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     4921 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     4981 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     5041 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     5101 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     5161 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     5221 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     5281 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     5341 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     5401 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     5461 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     5521 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     5581 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     5641 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     5701 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     5761 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     5821 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     5881 tccgactacg atgtggacgc tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     5941 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     6001 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     6061 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     6121 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     6181 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     6241 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     6301 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     6361 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     6421 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     6481 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     6541 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     6601 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     6661 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     6721 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     6781 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     6841 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     6901 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     6961 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     7021 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     7081 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     7141 cagaaacagc tgtttgtgga acagcacaag cactaccggg acgagatcat cgagcagatc
     7201 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     7261 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
     7321 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
     7381 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     7441 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc
     7501 acgaaaaagg ccggacaggc caaaaagaaa aagctcgagg gcggaggcgg gagcggatcc
     7561 ccctcccggc tccagatgtt cttcgctaat aaccacgacc aggaatttga ccctccaaag
     7621 gtttacccac ctgtcccagc tgagaagagg aagcccatcc gggtgctgtc tctctttgat
     7681 ggaatcgcta cagggctcct ggtgctgaag gacttgggca ttcaggtgga ccgctacatt
     7741 gcctcggagg tgtgtgagga ctccatcacg gtgggcatgg tgcggcacca ggggaagatc
     7801 atgtacgtcg gggacgtccg cagcgtcaca cagaagcata tccaggagtg gggcccattc
     7861 gatctggtga ttgggggcag tccctgcaat gacctctcca tcgtcaaccc tgctcgcaag
     7921 ggcctctacg agggcactgg ccggctcttc tttgagttct accgcctcct gcatgatgcg
     7981 cggcccaagg agggagatga tcgccccttc ttctggctct ttgagaatgt ggtggccatg
     8041 ggcgttagtg acaagaggga catctcgcga tttctcgagt ccaaccctgt gatgattgat
     8101 gccaaagaag tgtcagctgc acacagggcc cgctacttct ggggtaacct tcccggtatg
     8161 aacaggccgt tggcatccac tgtgaatgat aagctggagc tgcaggagtg tctggagcat
     8221 ggcaggatag ccaagttcag caaagtgagg accattacta cgaggtcaaa ctccataaag
     8281 cagggcaaag accagcattt tcctgtgttc atgaatgaga aagaggacat cttatggtgc
     8341 actgaaatgg aaagggtatt tggtttccca gtccactata ctgatgtgtc caacatgagc
     8401 cgcttggcga ggcagagact gctgggccgg tcatggagcg tgccagtcat ccgccacctc
     8461 ttcgctccgc tgaaggagta ttttgcgtgt gtgtccggcc ggcccggatc cggcgcaaca
     8521 aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc gaccgagtac
     8581 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     8641 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     8701 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     8761 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     8821 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     8881 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     8941 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     9001 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     9061 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     9121 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     9181 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     9241 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     9301 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     9361 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     9421 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     9481 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     9541 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     9601 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     9661 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     9721 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     9781 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     9841 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     9901 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     9961 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
    10021 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg
    10081 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
    10141 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
    10201 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
    10261 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
    10321 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
    10381 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
    10441 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
    10501 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
    10561 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
    10621 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
    10681 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
    10741 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
    10801 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
    10861 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
    10921 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
    10981 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
    11041 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
    11101 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
    11161 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
    11221 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
    11281 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
    11341 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
    11401 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
    11461 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
    11521 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
    11581 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
    11641 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
    11701 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
    11761 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
    11821 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
    11881 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
    11941 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
    12001 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
    12061 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
    12121 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
    12181 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
    12241 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
    12301 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
    12361 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
    12421 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
    12481 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
    12541 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
    12601 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
    12661 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
    12721 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
    12781 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
    12841 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
    12901 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
    12961 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
    13021 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
    13081 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
    13141 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
    13201 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
    13261 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
    13321 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
    13381 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
    13441 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
    13501 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
    13561 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
    13621 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
    13681 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
    13741 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
    13801 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
    13861 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
    13921 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
    13981 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
    14041 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ac
//
