LOCUS       sequence-255180-       16377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human codon optimized dCas9-Dnmt3a-Dnmt3l-T2A-eGFP + gRNA expression
            cassette + LV backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16377)
  AUTHORS   Tarjan DR, Flavahan WA, Bernstein BE
  TITLE     Epigenome editing strategies for the functional annotation of CTCF 
            insulators.
  JOURNAL   Nat Commun. 2019 Sep 18;10(1):4258. doi: 10.1038/s41467-019-12166-w.
  PUBMED    31534142
REFERENCE   2  (bases 1 to 16377)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-09-18"; volume: "10(1):4258. doi"; pages: " 
            10.1038/s41467-019-12166-w"
COMMENT     Sequence Label: pDT305
FEATURES             Location/Qualifiers
     source          1..16377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /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    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2364..2397)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_RNA        complement(2408..2483)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2500..2748)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2558..2577)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2728..2748)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        2781..3992
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3829..3848
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      4021..4030
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4030..4095
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4102..4122
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4132..4155,4156..4158,4159..6642,6643..6648,6649..6714,
                     6715..6717,6718..8232)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             7689..7697
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             8245..8265
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9958..9978
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9985..10038
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(10045..10047,10048..10758)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(10045..10047,10048..10758)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(10087..10108)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(10348..10367)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     10695..10716
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(10774..10807)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    10832..11420
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10885..10905)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11303..11314)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11423..11439)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11424..11440)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11945..12125
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(12151..12168)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12157..12381
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12427..12855
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12514..12533)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12724..12745
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12864..12884)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12869..13198
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      13049..13184
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     13111..13130
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        13246..13293
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             13312..13686
                     /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    13816..13937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13853..13872)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13907..13926
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13986..14002)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13986..14002)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13999..14021)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14010..14026
                     /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(14034..14040,14041..14058,14059..14064))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14079..14100
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14217..14234)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14388..14976)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14468..14487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15147..15938,15939..16007))
                     /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     15770..15789
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16008..16112)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(16187..16206)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaataactt cgtatagcat acattatacg aagttattaa
     2401 aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa
     2461 cttgctattt ctagctctaa aactgagacg tccgtctccg gtgtttcgtc ctttccacaa
     2521 gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat
     2581 tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt
     2641 attttgtact aatatctttg tgtttacagt caaattaatt ccaattatct ctctaacagc
     2701 cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt aattaattaa
     2761 cccgtgtcgg ctccagatct ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc
     2821 cctcctcacg gcgagcgctg ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc
     2881 cgcccggacg ctcaggacag cggcccgctg ctcataagac tcggccttag aaccccagta
     2941 tcagcagaag gacattttag gacgggactt gggtgactct agggcactgg ttttctttcc
     3001 agagagcgga acaggcgagg aaaagtagtc ccttctcggc gattctgcgg agggatctcc
     3061 gtggggcggt gaacgccgat gattatataa ggacgcgccg ggtgtggcac agctagttcc
     3121 gtcgcagccg ggatttgggt cgcggttctt gtttgtggat cgctgtgatc gtcacttggt
     3181 gagtagcggg ctgctgggct ggccggggct ttcgtggccg ccgggccgct cggtgggacg
     3241 gaagcgtgtg gagagaccgc caagggctgt agtctgggtc cgcgagcaag gttgccctga
     3301 actgggggtt ggggggagcg cagcaaaatg gcggctgttc ccgagtcttg aatggaagac
     3361 gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga
     3421 acccaaggtc ttgaggcctt cgctaatgcg ggaaagctct tattcgggtg agatgggctg
     3481 gggcaccatc tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg
     3541 tctgttgcgg gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga
     3601 gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg
     3661 gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc
     3721 tagggtaggc tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga
     3781 ggcgtcagtt tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt
     3841 tttgaactat gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg
     3901 aaatgtaatc atttgggtca atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta
     3961 aattctggcc gtttttggct tttttgttag acgaagcttg ggctgcaggt cgactctaga
     4021 gccaccatgg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag
     4081 gatgacgatg acaagatggc ccccaagaag aagaggaagg tgggccgcgg agacaagaag
     4141 tattcaatcg gcctggctat cggaaccaac agcgtggggt gggcagtcat cacagacgag
     4201 tataaggtcc cctccaagaa gtttaaggtg ctgggcaata ccgatcgcca ctccatcaag
     4261 aagaacctga tcggcgccct gctgttcgac tctggcgaga cagcagaggc aaccaggctg
     4321 aagaggacag cacggagaag gtatacccgc cggaagaacc ggatctgcta cctgcaggag
     4381 atcttcagca atgagatggc caaggtggac gattccttct ttcaccgcct ggaggagtct
     4441 ttcctggtgg aggaggataa gaagcacgag cggcacccaa tctttggcaa catcgtggac
     4501 gaggtggcct atcacgagaa gtaccccacc atctatcacc tgcggaagaa gctggtggat
     4561 tccacagaca aggccgatct gagactgatc tatctggccc tggcccacat gatcaagttc
     4621 aggggccact ttctgatcga gggcgacctg aatcccgata acagcgatgt ggacaagctg
     4681 ttcatccagc tggtgcagac ctacaaccag ctgtttgagg agaatcctat caacgcctct
     4741 ggagtggacg caaaggcaat cctgagcgcc agactgtcta agagcagaag gctggagaat
     4801 ctgatcgccc agctgcctgg cgagaagaag aatggcctgt ttggcaacct gatcgccctg
     4861 tccctgggcc tgaccccaaa tttcaagtct aactttgacc tggccgagga tgccaagctg
     4921 cagctgtcca aggatacata tgacgatgac ctggacaacc tgctggccca gatcggcgac
     4981 cagtacgccg atctgttcct ggccgccaag aacctgagcg atgccatcct gctgtccgac
     5041 atcctgcgcg tgaatacaga gatcaccaag gcccctctgt ccgcctctat gatcaagaga
     5101 tatgatgagc accaccagga cctgaccctg ctgaaggccc tggtgaggca gcagctgcca
     5161 gagaagtaca aggagatctt ctttgatcag agcaagaacg gatacgcagg atatatcgac
     5221 ggaggagcat cccaggagga gttctacaag tttatcaagc ctatcctgga gaagatggac
     5281 ggcaccgagg agctgctggt gaagctgaac cgggaggacc tgctgcggaa gcagagaaca
     5341 ttcgataatg gcagcatccc ccaccagatc cacctgggag agctgcacgc aatcctgcgc
     5401 cggcaggagg atttctaccc ttttctgaag gacaataggg agaagatcga gaagatcctg
     5461 accttccgca tcccatacta tgtgggacca ctggcaaggg gaaacagcag atttgcctgg
     5521 atgacaagaa agtccgagga gacaatcacc ccttggaatt tcgaggaggt ggtggataag
     5581 ggcgcctctg cccagagctt catcgagagg atgaccaact ttgacaagaa tctgcccaac
     5641 gagaaggtgc tgcctaagca ctctctgctg tacgagtatt tcacagtgta taatgagctg
     5701 accaaggtga agtacgtgac agagggcatg agaaagccag ccttcctgag cggcgagcag
     5761 aagaaggcca tcgtggacct gctgtttaag acaaacagga aggtgaccgt gaagcagctg
     5821 aaggaggact atttcaagaa gatcgagtgt tttgattccg tggagatctc tggcgtggag
     5881 gaccggttta atgcctccct gggcacatac cacgatctgc tgaagatcat caaggataag
     5941 gacttcctgg acaatgagga gaacgaggac atcctggagg atatcgtgct gacactgacc
     6001 ctgtttgagg atcgggagat gatcgaggag agactgaaga cctatgccca cctgttcgat
     6061 gacaaagtga tgaagcagct gaagagaagg cgctacacag gatggggcag gctgagccgc
     6121 aagctgatca acggcatcag agacaagcag tctggcaaga ccatcctgga ttttctgaag
     6181 agcgacggct tcgccaacag gaacttcatg cagctgatcc acgatgactc cctgacattc
     6241 aaggaggata tccagaaggc acaggtgtcc ggacagggcg actctctgca cgagcacatc
     6301 gccaatctgg ccggcagccc agccatcaag aagggcatcc tgcagaccgt gaaggtggtg
     6361 gacgagctgg tgaaagtgat gggcaggcac aagcccgaga atatcgtgat cgagatggcc
     6421 cgcgagaacc agaccacaca gaagggccag aagaatagca gggagcgcat gaagcgcatc
     6481 gaggagggca tcaaggagct gggctcccag atcctgaagg agcaccccgt ggagaatacc
     6541 cagctgcaga acgagaagct gtatctgtac tatctgcaga acggccggga catgtacgtg
     6601 gatcaggagc tggacatcaa tagactgtct gattatgacg tggccgccat cgtgccccag
     6661 tctttcctga aggatgacag catcgacaac aaggtgctga cacggagcga taaggccaga
     6721 ggcaagagcg acaatgtgcc ttccgaggag gtggtgaaga agatgaagaa ttactggcgg
     6781 cagctgctga acgccaagct gatcacccag cggaagtttg ataacctgac aaaggcagag
     6841 agaggaggcc tgtccgagct ggacaaggca ggcttcatca agaggcagct ggtggagaca
     6901 cgccagatca ccaagcacgt ggcccagatc ctggattctc gcatgaatac caagtacgac
     6961 gagaacgata agctgatccg ggaggtgaaa gtgatcaccc tgaagagcaa gctggtgtcc
     7021 gacttccgga aggacttcca gttttataag gtgcgcgaga tcaacaatta tcaccacgcc
     7081 cacgacgcat acctgaacgc agtggtggga accgccctga tcaagaagta cccaaagctg
     7141 gagagcgagt tcgtgtacgg cgattataag gtgtacgacg tgagaaagat gatcgccaag
     7201 tccgagcagg agatcggcaa ggccacagcc aagtatttct tttactctaa catcatgaac
     7261 ttctttaaga cagagatcac cctggcaaac ggagagatca ggaagcggcc cctgatcgag
     7321 acaaatggcg agacaggcga gatcgtgtgg gataagggcc gggactttgc caccgtgaga
     7381 aaggtgctga gcatgccaca ggtgaacatc gtgaagaaga cagaggtgca gaccggcggc
     7441 ttctccaagg agtctatcct gcccaagagg aattccgata agctgatcgc ccgcaagaag
     7501 gattgggacc ccaagaagta tggcggcttc gactccccta ccgtggccta ctctgtgctg
     7561 gtggtggcca aggtggagaa gggcaagtct aagaagctga agagcgtgaa ggagctgctg
     7621 ggcatcacaa tcatggagag gagctccttc gagaagaacc ccatcgattt tctggaggcc
     7681 aagggctata aggaggtgaa gaaggacctg atcatcaagc tgcctaagta ctccctgttt
     7741 gagctggaga atggaaggaa gaggatgctg gcatctgccg gagagctgca gaagggcaac
     7801 gagctggccc tgccaagcaa gtacgtgaat ttcctgtatc tggccagcca ctacgagaag
     7861 ctgaagggct cccccgagga taacgagcag aagcagctgt ttgtggagca gcacaagcac
     7921 tatctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgat
     7981 gccaacctgg acaaggtgct gtccgcctac aataagcaca gggacaagcc tatccgcgag
     8041 caggccgaga acatcatcca cctgttcaca ctgaccaatc tgggcgcccc agccgccttc
     8101 aagtattttg ataccacaat cgaccggaag agatacacat ctaccaagga ggtgctggat
     8161 gccaccctga tccaccagag catcacaggc ctgtacgaga caaggatcga cctgagccag
     8221 ctgggaggcg attcccgggc agatcccaag aagaagcgga aggtggaggc aagcggatcc
     8281 ggaagggcat ctcctggaat cccaggaagc acccgcaacc acgatcagga gtttgacccc
     8341 cctaaggtgt acccacccgt gccagccgag aagaggaagc ccatccgcgt gctgtccctg
     8401 ttcgacggca tcgccacagg cctgctggtg ctgaaggatc tgggcatcca ggtggacaga
     8461 tatatcgcct ccgaggtgtg cgaggattct atcaccgtgg gcatggtgag gcaccagggc
     8521 aagatcatgt acgtgggcga cgtgcgcagc gtgacacaga agcacatcca ggagtgggga
     8581 cccttcgacc tggtcatcgg aggcagcccc tgtaatgacc tgtccatcgt gaaccctgca
     8641 aggaagggcc tgtatgaggg aaccggcaga ctgttctttg agttctacag gctgctgcac
     8701 gacgcccgcc ctaaggaggg cgatgacagg ccattctttt ggctgtttga gaacgtggtg
     8761 gccatgggcg tgagcgacaa gcgggatatc tccagattcc tggagtctaa tcccgtgatg
     8821 atcgatgcaa aggaggtgtc tgccgcacac agggcaaggt acttttgggg aaatctgcct
     8881 ggcatgaacc gcccactggc cagcaccgtg aacgacaagc tggagctgca ggagtgcctg
     8941 gagcacggaa ggatcgccaa gttctccaag gtgcggacaa tcaccacaag atctaacagc
     9001 atcaagcagg gcaaggatca gcacttcccc gtgttcatga atgagaagga ggacatcctg
     9061 tggtgtaccg agatggagcg cgtgttcggc tttccagtgc actatacaga cgtgagcaat
     9121 atgagccggc tggcaaggca gagactgctg ggccggtcct ggtctgtgcc agtgatcaga
     9181 cacctgttcg cccccctgaa ggagtacttt gcctgcgtgt ctagcggcaa ctctaatgcc
     9241 aacagcagag gcccttcctt ttcctctggc ctggtgccac tgtctctgag gggcagccac
     9301 atgggcccca tggagatcta caagaccgtg tccgcctgga agaggcagcc tgtgcgcgtg
     9361 ctgtctctgt tccgcaacat cgacaaggtg ctgaagagcc tgggctttct ggagagcgga
     9421 tccggatctg gaggaggcac cctgaagtat gtggaggatg tgacaaatgt ggtgcggaga
     9481 gacgtggaga agtggggccc cttcgatctg gtgtacggat ccacccagcc actgggaagc
     9541 tcctgcgata ggtgtccagg atggtatatg ttccagtttc acagaatcct gcagtacgca
     9601 ctgccaaggc aggagagcca gcgccctttc ttttggatct ttatggacaa cctgctgctg
     9661 acagaggatg accaggagac aacaacccgc ttcctgcaga cagaggcagt gaccctgcag
     9721 gatgtgaggg gacgcgacta tcagaatgcc atgcgggtgt ggtctaacat ccctggcctg
     9781 aagagcaagc acgcccccct gacccctaag gaggaggagt acctgcaggc ccaggtgcgg
     9841 agcagatcca agctggatgc ccctaaggtg gaactgctgg tgaagaattg tctgctgcca
     9901 ctgcgggagt acttcaagta ctttagtcag aatagcctgc cactgtcacg agccgacccc
     9961 aagaagaaac ggaaggtgtc tagcgagggc agaggaagtc ttctaacatg cggtgacgtg
    10021 gaggagaatc ccggccctgg taccgtgagc aagggcgagg agctgttcac cggggtggtg
    10081 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
    10141 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
    10201 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
    10261 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
    10321 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
    10381 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
    10441 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
    10501 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
    10561 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
    10621 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
    10681 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
    10741 atggacgagc tgtacaagac cggttgataa tagataactt cgtatagcat acattatacg
    10801 aagttatgaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
    10861 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
    10921 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
    10981 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
    11041 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
    11101 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
    11161 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
    11221 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
    11281 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
    11341 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
    11401 cctttgggcc gcctccccgc atcgataccg tcgacctcga gacctagaaa aacatggagc
    11461 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga
    11521 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa
    11581 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
    11641 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc
    11701 tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg
    11761 ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac
    11821 ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga
    11881 gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg
    11941 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
    12001 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
    12061 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
    12121 tagcagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
    12181 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
    12241 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
    12301 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
    12361 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
    12421 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
    12481 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
    12541 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
    12601 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
    12661 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
    12721 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
    12781 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
    12841 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
    12901 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
    12961 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
    13021 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
    13081 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
    13141 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
    13201 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
    13261 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
    13321 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
    13381 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
    13441 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
    13501 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
    13561 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
    13621 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
    13681 gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
    13741 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
    13801 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
    13861 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
    13921 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
    13981 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
    14041 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
    14101 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
    14161 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
    14221 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
    14281 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
    14341 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
    14401 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
    14461 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
    14521 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
    14581 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
    14641 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
    14701 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
    14761 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
    14821 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
    14881 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
    14941 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
    15001 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
    15061 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
    15121 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
    15181 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
    15241 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
    15301 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
    15361 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
    15421 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
    15481 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
    15541 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
    15601 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
    15661 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
    15721 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
    15781 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
    15841 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
    15901 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
    15961 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
    16021 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
    16081 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
    16141 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
    16201 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
    16261 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
    16321 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
