LOCUS       sequence-249065-       16368 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 16368)
  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 16368)
  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..16368
                     /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"
     protein_bind    complement(2601..2634)
                     /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(2645..2720)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2737..2985)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2795..2814)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2965..2985)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        3018..4228
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4065..4084
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4255..4320
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4327..4347
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4357..4380,4381..4383,4384..6867,6868..6873,6874..6939,
                     6940..6942,6943..8457)
                     /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             7914..7922
                     /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             8470..8490
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10183..10203
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10213..10266
                     /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(10273..10275,10276..10986)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(10273..10275,10276..10986)
                     /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(10315..10336)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(10576..10595)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     10923..10944
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(11002..11035)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    11060..11648
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11113..11133)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11531..11542)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11651..11667)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11652..11668)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12173..12353
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(12379..12396)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12385..12609
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12655..13083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12742..12761)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12952..12973
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(13092..13112)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        13097..13426
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      13277..13412
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     13339..13358
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        13474..13521
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             13540..13914
                     /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    14044..14165
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(14081..14100)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     14135..14154
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(14214..14230)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14214..14230)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14227..14249)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14238..14254
                     /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(14262..14268,14269..14286,14287..14292))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14307..14328
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14445..14462)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14616..15204)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14696..14715)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15375..16166,16167..16235))
                     /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     15998..16017
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16236..16340)
                     /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 ataacttcgt atagcataca ttatacgaag ttattaaaaa
     2641 aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt
     2701 gctatttcta gctctaaaac tgagacgtcc gtctccggtg tttcgtcctt tccacaagat
     2761 atataaagcc aagaaatcga aatactttca agttacggta agcatatgat agtccatttt
     2821 aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt
     2881 ttgtactaat atctttgtgt ttacagtcaa attaattcca attatctctc taacagcctt
     2941 gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcttaat taattaaccc
     3001 gtgtcggctc cagatctggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct
     3061 cctcacggcg agcgctgcca cgtcagacga agggcgcagc gagcgtcctg atccttccgc
     3121 ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca
     3181 gcagaaggac attttaggac gggacttggg tgactctagg gcactggttt tctttccaga
     3241 gagcggaaca ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg
     3301 gggcggtgaa cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc
     3361 gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag
     3421 tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggaa
     3481 gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact
     3541 gggggttggg gggagcgcac aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt
     3601 gtaaggcggg ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc
     3661 aaggtcttga ggccttcgct aatgcgggaa agctcttatt cgggtgagat gggctggggc
     3721 accatctggg gaccctgacg tgaagtttgt cactgactgg agaactcggg tttgtcgtct
     3781 ggttgcgggg gcggcagtta tgcggtgccg ttgggcagtg cacccgtacc tttgggagcg
     3841 cgcgcctcgt cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca
     3901 cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg
     3961 gtaggctctc ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg
     4021 tcagtttctt tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg
     4081 aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat
     4141 gtaatcattt gggtcaatat gtaattttca gtgttagact agtaaattgt ccgctaaatt
     4201 ctggccgttt ttggcttttt tgttagacga agcttgggct gcaggtcgac tatggactac
     4261 aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag
     4321 atggccccca agaagaagag gaaggtgggc cgcggagaca agaagtattc aatcggcctg
     4381 gctatcggaa ccaacagcgt ggggtgggca gtcatcacag acgagtataa ggtcccctcc
     4441 aagaagttta aggtgctggg caataccgat cgccactcca tcaagaagaa cctgatcggc
     4501 gccctgctgt tcgactctgg cgagacagca gaggcaacca ggctgaagag gacagcacgg
     4561 agaaggtata cccgccggaa gaaccggatc tgctacctgc aggagatctt cagcaatgag
     4621 atggccaagg tggacgattc cttctttcac cgcctggagg agtctttcct ggtggaggag
     4681 gataagaagc acgagcggca cccaatcttt ggcaacatcg tggacgaggt ggcctatcac
     4741 gagaagtacc ccaccatcta tcacctgcgg aagaagctgg tggattccac agacaaggcc
     4801 gatctgagac tgatctatct ggccctggcc cacatgatca agttcagggg ccactttctg
     4861 atcgagggcg acctgaatcc cgataacagc gatgtggaca agctgttcat ccagctggtg
     4921 cagacctaca accagctgtt tgaggagaat cctatcaacg cctctggagt ggacgcaaag
     4981 gcaatcctga gcgccagact gtctaagagc agaaggctgg agaatctgat cgcccagctg
     5041 cctggcgaga agaagaatgg cctgtttggc aacctgatcg ccctgtccct gggcctgacc
     5101 ccaaatttca agtctaactt tgacctggcc gaggatgcca agctgcagct gtccaaggat
     5161 acatatgacg atgacctgga caacctgctg gcccagatcg gcgaccagta cgccgatctg
     5221 ttcctggccg ccaagaacct gagcgatgcc atcctgctgt ccgacatcct gcgcgtgaat
     5281 acagagatca ccaaggcccc tctgtccgcc tctatgatca agagatatga tgagcaccac
     5341 caggacctga ccctgctgaa ggccctggtg aggcagcagc tgccagagaa gtacaaggag
     5401 atcttctttg atcagagcaa gaacggatac gcaggatata tcgacggagg agcatcccag
     5461 gaggagttct acaagtttat caagcctatc ctggagaaga tggacggcac cgaggagctg
     5521 ctggtgaagc tgaaccggga ggacctgctg cggaagcaga gaacattcga taatggcagc
     5581 atcccccacc agatccacct gggagagctg cacgcaatcc tgcgccggca ggaggatttc
     5641 tacccttttc tgaaggacaa tagggagaag atcgagaaga tcctgacctt ccgcatccca
     5701 tactatgtgg gaccactggc aaggggaaac agcagatttg cctggatgac aagaaagtcc
     5761 gaggagacaa tcaccccttg gaatttcgag gaggtggtgg ataagggcgc ctctgcccag
     5821 agcttcatcg agaggatgac caactttgac aagaatctgc ccaacgagaa ggtgctgcct
     5881 aagcactctc tgctgtacga gtatttcaca gtgtataatg agctgaccaa ggtgaagtac
     5941 gtgacagagg gcatgagaaa gccagccttc ctgagcggcg agcagaagaa ggccatcgtg
     6001 gacctgctgt ttaagacaaa caggaaggtg accgtgaagc agctgaagga ggactatttc
     6061 aagaagatcg agtgttttga ttccgtggag atctctggcg tggaggaccg gtttaatgcc
     6121 tccctgggca cataccacga tctgctgaag atcatcaagg ataaggactt cctggacaat
     6181 gaggagaacg aggacatcct ggaggatatc gtgctgacac tgaccctgtt tgaggatcgg
     6241 gagatgatcg aggagagact gaagacctat gcccacctgt tcgatgacaa agtgatgaag
     6301 cagctgaaga gaaggcgcta cacaggatgg ggcaggctga gccgcaagct gatcaacggc
     6361 atcagagaca agcagtctgg caagaccatc ctggattttc tgaagagcga cggcttcgcc
     6421 aacaggaact tcatgcagct gatccacgat gactccctga cattcaagga ggatatccag
     6481 aaggcacagg tgtccggaca gggcgactct ctgcacgagc acatcgccaa tctggccggc
     6541 agcccagcca tcaagaaggg catcctgcag accgtgaagg tggtggacga gctggtgaaa
     6601 gtgatgggca ggcacaagcc cgagaatatc gtgatcgaga tggcccgcga gaaccagacc
     6661 acacagaagg gccagaagaa tagcagggag cgcatgaagc gcatcgagga gggcatcaag
     6721 gagctgggct cccagatcct gaaggagcac cccgtggaga atacccagct gcagaacgag
     6781 aagctgtatc tgtactatct gcagaacggc cgggacatgt acgtggatca ggagctggac
     6841 atcaatagac tgtctgatta tgacgtggcc gccatcgtgc cccagtcttt cctgaaggat
     6901 gacagcatcg acaacaaggt gctgacacgg agcgataagg ccagaggcaa gagcgacaat
     6961 gtgccttccg aggaggtggt gaagaagatg aagaattact ggcggcagct gctgaacgcc
     7021 aagctgatca cccagcggaa gtttgataac ctgacaaagg cagagagagg aggcctgtcc
     7081 gagctggaca aggcaggctt catcaagagg cagctggtgg agacacgcca gatcaccaag
     7141 cacgtggccc agatcctgga ttctcgcatg aataccaagt acgacgagaa cgataagctg
     7201 atccgggagg tgaaagtgat caccctgaag agcaagctgg tgtccgactt ccggaaggac
     7261 ttccagtttt ataaggtgcg cgagatcaac aattatcacc acgcccacga cgcatacctg
     7321 aacgcagtgg tgggaaccgc cctgatcaag aagtacccaa agctggagag cgagttcgtg
     7381 tacggcgatt ataaggtgta cgacgtgaga aagatgatcg ccaagtccga gcaggagatc
     7441 ggcaaggcca cagccaagta tttcttttac tctaacatca tgaacttctt taagacagag
     7501 atcaccctgg caaacggaga gatcaggaag cggcccctga tcgagacaaa tggcgagaca
     7561 ggcgagatcg tgtgggataa gggccgggac tttgccaccg tgagaaaggt gctgagcatg
     7621 ccacaggtga acatcgtgaa gaagacagag gtgcagaccg gcggcttctc caaggagtct
     7681 atcctgccca agaggaattc cgataagctg atcgcccgca agaaggattg ggaccccaag
     7741 aagtatggcg gcttcgactc ccctaccgtg gcctactctg tgctggtggt ggccaaggtg
     7801 gagaagggca agtctaagaa gctgaagagc gtgaaggagc tgctgggcat cacaatcatg
     7861 gagaggagct ccttcgagaa gaaccccatc gattttctgg aggccaaggg ctataaggag
     7921 gtgaagaagg acctgatcat caagctgcct aagtactccc tgtttgagct ggagaatgga
     7981 aggaagagga tgctggcatc tgccggagag ctgcagaagg gcaacgagct ggccctgcca
     8041 agcaagtacg tgaatttcct gtatctggcc agccactacg agaagctgaa gggctccccc
     8101 gaggataacg agcagaagca gctgtttgtg gagcagcaca agcactatct ggacgagatc
     8161 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgatgccaa cctggacaag
     8221 gtgctgtccg cctacaataa gcacagggac aagcctatcc gcgagcaggc cgagaacatc
     8281 atccacctgt tcacactgac caatctgggc gccccagccg ccttcaagta ttttgatacc
     8341 acaatcgacc ggaagagata cacatctacc aaggaggtgc tggatgccac cctgatccac
     8401 cagagcatca caggcctgta cgagacaagg atcgacctga gccagctggg aggcgattcc
     8461 cgggcagatc ccaagaagaa gcggaaggtg gaggcaagcg gatccggaag ggcatctcct
     8521 ggaatcccag gaagcacccg caaccacgat caggagtttg acccccctaa ggtgtaccca
     8581 cccgtgccag ccgagaagag gaagcccatc cgcgtgctgt ccctgttcga cggcatcgcc
     8641 acaggcctgc tggtgctgaa ggatctgggc atccaggtgg acagatatat cgcctccgag
     8701 gtgtgcgagg attctatcac cgtgggcatg gtgaggcacc agggcaagat catgtacgtg
     8761 ggcgacgtgc gcagcgtgac acagaagcac atccaggagt ggggaccctt cgacctggtc
     8821 atcggaggca gcccctgtaa tgacctgtcc atcgtgaacc ctgcaaggaa gggcctgtat
     8881 gagggaaccg gcagactgtt ctttgagttc tacaggctgc tgcacgacgc ccgccctaag
     8941 gagggcgatg acaggccatt cttttggctg tttgagaacg tggtggccat gggcgtgagc
     9001 gacaagcggg atatctccag attcctggag tctaatcccg tgatgatcga tgcaaaggag
     9061 gtgtctgccg cacacagggc aaggtacttt tggggaaatc tgcctggcat gaaccgccca
     9121 ctggccagca ccgtgaacga caagctggag ctgcaggagt gcctggagca cggaaggatc
     9181 gccaagttct ccaaggtgcg gacaatcacc acaagatcta acagcatcaa gcagggcaag
     9241 gatcagcact tccccgtgtt catgaatgag aaggaggaca tcctgtggtg taccgagatg
     9301 gagcgcgtgt tcggctttcc agtgcactat acagacgtga gcaatatgag ccggctggca
     9361 aggcagagac tgctgggccg gtcctggtct gtgccagtga tcagacacct gttcgccccc
     9421 ctgaaggagt actttgcctg cgtgtctagc ggcaactcta atgccaacag cagaggccct
     9481 tccttttcct ctggcctggt gccactgtct ctgaggggca gccacatggg ccccatggag
     9541 atctacaaga ccgtgtccgc ctggaagagg cagcctgtgc gcgtgctgtc tctgttccgc
     9601 aacatcgaca aggtgctgaa gagcctgggc tttctggaga gcggatccgg atctggagga
     9661 ggcaccctga agtatgtgga ggatgtgaca aatgtggtgc ggagagacgt ggagaagtgg
     9721 ggccccttcg atctggtgta cggatccacc cagccactgg gaagctcctg cgataggtgt
     9781 ccaggatggt atatgttcca gtttcacaga atcctgcagt acgcactgcc aaggcaggag
     9841 agccagcgcc ctttcttttg gatctttatg gacaacctgc tgctgacaga ggatgaccag
     9901 gagacaacaa cccgcttcct gcagacagag gcagtgaccc tgcaggatgt gaggggacgc
     9961 gactatcaga atgccatgcg ggtgtggtct aacatccctg gcctgaagag caagcacgcc
    10021 cccctgaccc ctaaggagga ggagtacctg caggcccagg tgcggagcag atccaagctg
    10081 gatgccccta aggtggacct gctggtgaag aattgtctgc tgccactgcg ggagtacttc
    10141 aagtacttta gtcagaatag cctgccactg tcacgagccg accccaagaa gaaacggaag
    10201 gtgtctagaa gcgagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc
    10261 ggccctggta ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
    10321 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
    10381 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
    10441 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
    10501 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
    10561 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
    10621 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
    10681 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
    10741 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
    10801 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
    10861 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
    10921 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
    10981 tacaagaccg gttgataata gataacttcg tatagcatac attatacgaa gttatgaatt
    11041 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
    11101 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
    11161 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
    11221 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
    11281 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
    11341 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
    11401 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
    11461 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
    11521 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
    11581 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
    11641 ctccccgcat cgataccgtc gacctcgaga cctagaaaaa catggagcaa tcacaagtag
    11701 caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt
    11761 gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga
    11821 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
    11881 acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa
    11941 ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct acaagctagt
    12001 accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc gcttgttaca
    12061 ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt ggaggtttga
    12121 cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta ctgggtctct
    12181 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
    12241 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
    12301 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagggcccg
    12361 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
    12421 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
    12481 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
    12541 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
    12601 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
    12661 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
    12721 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
    12781 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
    12841 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
    12901 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
    12961 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
    13021 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
    13081 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
    13141 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
    13201 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
    13261 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
    13321 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
    13381 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
    13441 ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat
    13501 atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc
    13561 gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc
    13621 gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc
    13681 ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg
    13741 gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg
    13801 gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc
    13861 ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg
    13921 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
    13981 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
    14041 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
    14101 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
    14161 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
    14221 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
    14281 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
    14341 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
    14401 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
    14461 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
    14521 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
    14581 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
    14641 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
    14701 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
    14761 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
    14821 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
    14881 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
    14941 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
    15001 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
    15061 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
    15121 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
    15181 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
    15241 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
    15301 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
    15361 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
    15421 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
    15481 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
    15541 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
    15601 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
    15661 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
    15721 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
    15781 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
    15841 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
    15901 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
    15961 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
    16021 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
    16081 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
    16141 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
    16201 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
    16261 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
    16321 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgac
//
