LOCUS       sequence-441134-       14634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EFS promoter driven human codon optimized SpdCas9 directly
            fused to WT Dnmt3A/3L followed by P2A-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14634)
  AUTHORS   Guerra-Resendez RS, Lydon SL, Ma AJ, Bedford GC, Reed DR, Kim S, 
            Teran ER, Nishiguchi T, Escobar M, DiNardo AR, Hilton IB
  TITLE     Characterization of Rationally Designed CRISPR/Cas9-Based DNA 
            Methyltransferases with Distinct Methyltransferase and Gene 
            Silencing Activities in Human Cell Lines and Primary Human T Cells.
  JOURNAL   ACS Synth Biol. 2025 Feb 3. doi: 10.1021/acssynbio.4c00569.
  PUBMED    39898483
REFERENCE   2  (bases 1 to 14634)
  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: "ACS Synth 
            Biol. 2025 Feb 3. doi: 10.1021/acssynbio.4c00569."
COMMENT     Sequence Label: EFS-SpdCas9-Dnmt3A/3L-WT
FEATURES             Location/Qualifiers
     source          1..14634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(53..72)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(732..1523,1524..1592))
                     /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     1355..1374
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1593..1697)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1772..1791)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1963..2342
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2344..2542
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2497..2517
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2560..2740
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2787..2912
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3405..3638
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3823..3867
                     /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    4165..4282
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        4414..4489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4551..4762
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4776..4785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4782..4808,4809..4811,4812..7298,7299..7301,
                     7302..8885)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4782..8885
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             8886..8933
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8946..8963
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             8973..8990
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             10635..10658
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10668..10724
                     /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             10725..11435
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(10870..10888)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     11138..11157
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    11451..12039
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11504..11524)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11922..11933)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12111..12344
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12370..12387)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12376..12600
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      12646..13074
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12733..12752)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12943..12964
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(13083..13103)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        13088..13417
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      13268..13403
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     13330..13349
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        13465..13512
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             13531..13905
                     /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    14035..14156
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(14072..14091)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     14126..14145
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(14205..14221)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14205..14221)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14218..14240)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14229..14245
                     /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(14253..14259,14260..14277,14278..14283))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14298..14319
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14436..14453)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(14607..14634,1..561))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
       61 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
      121 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
      181 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
      241 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
      301 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
      361 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
      421 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
      481 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
      541 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
      601 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
      661 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
      721 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
      781 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
      841 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
      901 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
      961 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     1021 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     1081 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     1141 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     1201 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     1261 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     1321 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     1381 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     1441 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     1501 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     1561 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     1621 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     1681 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     1741 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     1801 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     1861 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     1921 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     1981 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     2041 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     2101 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     2161 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     2221 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     2281 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     2341 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     2401 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     2461 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     2521 ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag
     2581 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     2641 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     2701 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
     2761 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
     2821 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
     2881 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     2941 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     3001 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     3061 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     3121 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     3181 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     3241 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     3301 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     3361 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     3421 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     3481 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     3541 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     3601 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     3661 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     3721 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     3781 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     3841 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     3901 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     3961 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     4021 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggatagaga
     4081 gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag
     4141 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     4201 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     4261 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt
     4321 aattaaggta ccgatatcac tagtgttcca ttaactgcgc taaaaataat ttttaaatct
     4381 tttttagctt cttgctcttt tttgtacgtc tctgttttag agctagaaat agcaagttaa
     4441 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgaatt
     4501 cgctagctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg
     4561 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat ccggtgccta
     4621 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     4681 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     4741 cgggtttgcc gccagaacac aggaccggtt ctagagccac catggacaag aagtacagca
     4801 tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     4861 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     4921 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     4981 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     5041 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     5101 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     5161 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     5221 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     5281 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     5341 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     5401 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     5461 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     5521 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     5581 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     5641 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     5701 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     5761 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     5821 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     5881 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     5941 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     6001 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     6061 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     6121 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     6181 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     6241 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     6301 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     6361 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     6421 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     6481 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     6541 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     6601 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     6661 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     6721 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     6781 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     6841 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     6901 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     6961 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     7021 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     7081 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     7141 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     7201 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     7261 aactggacat caaccggctg tccgactacg atgtggacgc catcgtgcct cagagctttc
     7321 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     7381 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     7441 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     7501 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     7561 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     7621 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     7681 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     7741 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     7801 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     7861 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     7921 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     7981 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     8041 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     8101 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     8161 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     8221 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     8281 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     8341 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     8401 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     8461 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     8521 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     8581 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     8641 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     8701 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     8761 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     8821 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     8881 gcgacaagcg acctgccgcc acaaagaagg ctggacaggc taagaagaag aaaatgggca
     8941 gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatgaacc
     9001 atgaccagga atttgacccc ccaaaggttt acccacctgt gccagctgag aagaggaagc
     9061 ccatccgcgt gctgtctctc tttgatggga ttgctacagg gctcctggtg ctgaaggacc
     9121 tgggcatcca agtggaccgc tacattgcct ccgaggtgtg tgaggactcc atcacggtgg
     9181 gcatggtgcg gcaccaggga aagatcatgt acgtcgggga cgtccgcagc gtcacacaga
     9241 agcatatcca ggagtggggc ccattcgacc tggtgattgg aggcagtccc tgcaatgacc
     9301 tctccattgt caaccctgcc cgcaagggac tttatgaggg tactggccgc ctcttctttg
     9361 agttctaccg cctcctgcat gatgcgcggc ccaaggaggg agatgatcgc cccttcttct
     9421 ggctctttga gaatgtggtg gccatgggcg ttagtgacaa gagggacatc tcgcgatttc
     9481 ttgagtctaa ccccgtgatg attgacgcca aagaagtgtc tgctgcacac agggcccgtt
     9541 acttctgggg taaccttcct ggcatgaaca ggcctttggc atccactgtg aatgataagc
     9601 tggagctgca agagtgtctg gagcacggca gaatagccaa gttcagcaaa gtgaggacca
     9661 ttaccaccag gtcaaactct ataaagcagg gcaaagacca gcatttcccc gtcttcatga
     9721 acgagaagga ggacatcctg tggtgcactg aaatggaaag ggtgtttggc ttccccgtcc
     9781 actacacaga cgtctccaac atgagccgct tggcgaggca gagactgctg ggccgatcgt
     9841 ggagcgtgcc ggtcatccgc cacctcttcg ctccgctgaa ggaatatttt gcttgtgtgt
     9901 ctagcggcaa tagtaacgct aacagccgcg ggccgagctt cagcagcggc ctggtgccgt
     9961 taagcttgcg cggcagccat atgggcccta tggagatata caagacagtg tctgcatgga
    10021 agagacagcc agtgcgggta ctgagccttt ttagaaatat tgataaagta ctaaagagtt
    10081 tgggcttttt agaaagcggt tctggttctg ggggaggaac gctgaagtac gtggaagatg
    10141 tcacaaatgt cgtgaggaga gacgtggaga aatggggccc ctttgacctg gtgtacggct
    10201 cgacgcagcc cctaggcagc tcctgtgatc gctgtcccgg ctggtacatg ttccagttcc
    10261 accggatcct gcagtatgcg ctgcctcgcc aggagagtca gcggcccttc ttctggatat
    10321 tcatggacaa tctgctgctg actgaggatg accaagagac aactacccgc ttccttcaga
    10381 cagaggctgt gaccctccag gatgtccgtg gcagggacta ccagaatgct atgcgggtgt
    10441 ggagcaacat tccagggctg aagagcaagc atgcgcccct gaccccaaag gaagaagagt
    10501 atctgcaagc ccaagtcaga agcaggagca agctggacgc cccgaaagtt gacctcctgg
    10561 tgaagaactg ccttctcccg ctgagagagt acttcaagta tttttctcaa aactcacttc
    10621 ctcttgctgg atccgattac aaagacgatg acgataaggg atccggcgca acaaacttct
    10681 ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accgatggtg agcaagggcg
    10741 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
    10801 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
    10861 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
    10921 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
    10981 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
    11041 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
    11101 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
    11161 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
    11221 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
    11281 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
    11341 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
    11401 actccaccgg cggcatggac gagctgtaca agtagtgtcg ttaagtcgac aatcaacctc
    11461 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
    11521 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
    11581 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
    11641 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
    11701 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
    11761 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
    11821 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
    11881 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
    11941 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
    12001 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
    12061 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
    12121 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
    12181 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
    12241 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
    12301 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc
    12361 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
    12421 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
    12481 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
    12541 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
    12601 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
    12661 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
    12721 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
    12781 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
    12841 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
    12901 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
    12961 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
    13021 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
    13081 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
    13141 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
    13201 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
    13261 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
    13321 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
    13381 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
    13441 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
    13501 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
    13561 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
    13621 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
    13681 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
    13741 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
    13801 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
    13861 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
    13921 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
    13981 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
    14041 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
    14101 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
    14161 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
    14221 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
    14281 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
    14341 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
    14401 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
    14461 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
    14521 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
    14581 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgag
//
