LOCUS       sequence-441221-       15076 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses TRE3GV promoter driven human codon optimized SpdCas9 
            directly fused to WT Dnmt3A/3L and hPGK promoter driven mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15076)
  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 15076)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: TRE3GV-SpdCas9-Dnmt3A/3L-WT
FEATURES             Location/Qualifiers
     source          1..15076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(47..635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(127..146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(806..1597,1598..1666))
                     /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     1429..1448
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1667..1771)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1846..1865)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2037..2416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2418..2616
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             2634..2814
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2861..2986
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3479..3712
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3897..3941
                     /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    4239..4356
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4457..4806
                     /label=TRE3GV promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, optimized for 
                     retroviral and lentiviral vectors"
     protein_bind    4465..4483
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4501..4519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4537..4555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4573..4591
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4609..4627
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4645..4663
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4681..4699
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4745..4769
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      4822..4831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4828..4854,4855..4857,4858..7344,7345..7347,
                     7348..8931)
                     /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             4828..8931
                     /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             8932..8979
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10627..10650
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(10686..10719)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        10720..11230
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     11149..11167
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      11234..11243
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             11240..11950
                     /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(11385..11403)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     11653..11672
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    11967..12555
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12020..12040)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12438..12449)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12627..12860
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12886..12903)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12892..13116
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      13162..13590
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13249..13268)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13459..13480
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(13599..13619)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        13604..13933
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      13784..13919
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     13846..13865
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        13981..14028
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             14047..14421
                     /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    14551..14672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(14588..14607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     14642..14661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(14721..14737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14721..14737)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14734..14756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14745..14761
                     /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(14769..14775,14776..14793,14794..14799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14814..14835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14952..14969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
       61 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
      121 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
      181 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
      241 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
      301 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
      361 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
      421 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
      481 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
      541 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
      601 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
      661 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
      721 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
      781 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
      841 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
      901 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
      961 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     1021 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     1081 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     1141 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     1201 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     1261 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     1321 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     1381 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     1441 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     1501 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     1561 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     1621 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     1681 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     1741 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     1801 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     1861 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     1921 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     1981 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     2041 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     2101 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     2161 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     2221 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     2281 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     2341 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     2401 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     2461 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     2521 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     2581 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
     2641 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     2701 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     2761 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     2821 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     2881 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     2941 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     3001 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     3061 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     3121 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     3181 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     3241 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     3301 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
     3361 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     3421 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     3481 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     3541 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     3601 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     3661 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     3721 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     3781 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     3841 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     3901 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     3961 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     4021 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     4081 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag
     4141 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg
     4201 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     4261 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     4321 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     4381 gatccagttt ggttaattaa ggtaccgcta gcatcgatat taaggcttat gcgatgaggc
     4441 cctttcgtct tcactcgagt ttactcccta tcagtgatag agaacgtatg aagagtttac
     4501 tccctatcag tgatagagaa cgtatgcaga ctttactccc tatcagtgat agagaacgta
     4561 taaggagttt actccctatc agtgatagag aacgtatgac cagtttactc cctatcagtg
     4621 atagagaacg tatctacagt ttactcccta tcagtgatag agaacgtata tccagtttac
     4681 tccctatcag tgatagagaa cgtatgtcga ggtaggcgtg tacggtgggc gcctataaaa
     4741 gcagagctcg tttagtgaac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt
     4801 atactttgga gaattgctag cgccaccatg gacaagaagt acagcatcgg cctggccatc
     4861 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     4921 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     4981 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     5041 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     5101 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     5161 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     5221 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     5281 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     5341 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     5401 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     5461 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     5521 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     5581 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     5641 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     5701 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     5761 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     5821 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     5881 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     5941 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     6001 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     6061 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     6121 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     6181 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     6241 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     6301 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     6361 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     6421 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     6481 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     6541 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     6601 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     6661 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     6721 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     6781 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     6841 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     6901 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     6961 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     7021 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     7081 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     7141 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     7201 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     7261 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     7321 cggctgtccg actacgatgt ggacgccatc gtgcctcaga gctttctgaa ggacgactcc
     7381 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     7441 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     7501 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     7561 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     7621 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     7681 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     7741 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     7801 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     7861 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     7921 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     7981 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     8041 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     8101 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     8161 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     8221 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     8281 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     8341 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     8401 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     8461 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     8521 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     8581 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     8641 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     8701 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     8761 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     8821 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     8881 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caagcgacct
     8941 gccgccacaa agaaggctgg acaggctaag aagaagaaag gaagcatgaa ccatgaccag
     9001 gaatttgacc ccccaaaggt ttacccacct gtgccagctg agaagaggaa gcccatccgc
     9061 gtgctgtctc tctttgatgg gattgctaca gggctcctgg tgctgaagga cctgggcatc
     9121 caagtggacc gctacattgc ctccgaggtg tgtgaggact ccatcacggt gggcatggtg
     9181 cggcaccagg gaaagatcat gtacgtcggg gacgtccgca gcgtcacaca gaagcatatc
     9241 caggagtggg gcccattcga cctggtgatt ggaggcagtc cctgcaatga cctctccatt
     9301 gtcaaccctg cccgcaaggg actttatgag ggtactggcc gcctcttctt tgagttctac
     9361 cgcctcctgc atgatgcgcg gcccaaggag ggagatgatc gccccttctt ctggctcttt
     9421 gagaatgtgg tggccatggg cgttagtgac aagagggaca tctcgcgatt tcttgagtct
     9481 aaccccgtga tgattgacgc caaagaagtg tctgctgcac acagggcccg ttacttctgg
     9541 ggtaaccttc ctggcatgaa caggcctttg gcatccactg tgaatgataa gctggagctg
     9601 caagagtgtc tggagcacgg cagaatagcc aagttcagca aagtgaggac cattaccacc
     9661 aggtcaaact ctataaagca gggcaaagac cagcatttcc ccgtcttcat gaacgagaag
     9721 gaggacatcc tgtggtgcac tgaaatggaa agggtgtttg gcttccccgt ccactacaca
     9781 gacgtctcca acatgagccg cttggcgagg cagagactgc tgggccgatc gtggagcgtg
     9841 ccggtcatcc gccacctctt cgctccgctg aaggaatatt ttgcttgtgt gtctagcggc
     9901 aatagtaacg ctaacagccg cgggccgagc ttcagcagcg gcctggtgcc gttaagcttg
     9961 cgcggcagcc atatgggccc tatggagata tacaagacag tgtctgcatg gaagagacag
    10021 ccagtgcggg tactgagcct ttttagaaat attgataaag tactaaagag tttgggcttt
    10081 ttagaaagcg gttctggttc tgggggagga acgctgaagt acgtggaaga tgtcacaaat
    10141 gtcgtgagga gagacgtgga gaaatggggc ccctttgacc tggtgtacgg ctcgacgcag
    10201 cccctaggca gctcctgtga tcgctgtccc ggctggtaca tgttccagtt ccaccggatc
    10261 ctgcagtatg cgctgcctcg ccaggagagt cagcggccct tcttctggat attcatggac
    10321 aatctgctgc tgactgagga tgaccaagag acaactaccc gcttccttca gacagaggct
    10381 gtgaccctcc aggatgtccg tggcagggac taccagaatg ctatgcgggt gtggagcaac
    10441 attccagggc tgaagagcaa gcatgcgccc ctgaccccaa aggaagaaga gtatctgcaa
    10501 gcccaagtca gaagcaggag caagctggac gccccgaaag ttgacctcct ggtgaagaac
    10561 tgccttctcc cgctgagaga gtacttcaag tatttttctc aaaactcact tcctcttgct
    10621 ggatccgatt acaaagacga tgacgataag ggatcttgat catctcaaca ctgacttgag
    10681 gatccataac ttcgtatagc atacattata cgaagttatg gggttggggt tgcgcctttt
    10741 ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg
    10801 cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat
    10861 cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga
    10921 aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag
    10981 taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt
    11041 ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg
    11101 ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt
    11161 ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct
    11221 ctctccccag caagccacca tggttagcaa gggcgaggag gataacatgg ccatcatcaa
    11281 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
    11341 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
    11401 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
    11461 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
    11521 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
    11581 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
    11641 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
    11701 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
    11761 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
    11821 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
    11881 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
    11941 gtacaagtag gaattcactc gaggacaatc aacctctgga ttacaaaatt tgtgaaagat
    12001 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
    12061 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
    12121 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
    12181 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
    12241 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
    12301 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
    12361 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
    12421 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
    12481 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
    12541 gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag
    12601 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
    12661 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
    12721 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
    12781 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
    12841 agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg actgtgcctt
    12901 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
    12961 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
    13021 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
    13081 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
    13141 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
    13201 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
    13261 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
    13321 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
    13381 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
    13441 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
    13501 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
    13561 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
    13621 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
    13681 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
    13741 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
    13801 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
    13861 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
    13921 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
    13981 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
    14041 taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
    14101 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
    14161 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
    14221 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
    14281 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
    14341 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
    14401 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat
    14461 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
    14521 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
    14581 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
    14641 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
    14701 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
    14761 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
    14821 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
    14881 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
    14941 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
    15001 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
    15061 aacatgtgag caaaag
//
