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TbVk|ePuSft?FE q N"U"7#$0%%&&}''e() G 6XwCNJZR<n!!!s"#%
&"&f&&&'~(()D 6 ` 56t
\]Ujrstuv{yXY;>T"6#$)$z$$S%%&!&&&&&|''}(  :J  GACNNNNNNGTC,CCAGGCCTGG,GATCCGGATC,CCWGGTACCWGG,CCANNNNNTGG,CCAGGNNNTGG,CCANNNCCTGG,CCAGGNCCTGG,GCACNNNAACGTTNNNGTGC,CACNNNGTG,CCNNNNNNNGG,AACNAACRYGTGCGTGC,GCACGCACRYGTTNGTT,GCACNAACRYGTTNGTGC,AACGCACRYGTGCGTT,GCACNNNNNNGTTSAACNNNNNNGTGC,GACNNNNNGTC,GAANNNNNNNTTGG,CCAANNNNNNNTTC,CACNNNNGTG,AACACNNNNGTGC,GCACNNNNGTGTT,GCANNNNNNTGC,GAACNNNNNNTCC,GGANNNNNNGTTC,CAGNNNCTG,CCAGGNNCTG,CAGNNCCTGG,CCWGGGCCC,GGGCCCWGG,CCWGGGCCCWGG,AACNNNNNNGTGCACNNNNNNGTT,GACNNNNNNTTYG,CRAANNNNNNGTC,GACNNNGTC,GAANNNNTTC,CCWGGTACC,GGTACCWGG,CCWGGWCCWGG,ACNNNNGTAYC,GRTACNNNNGT,CCWGGYRCCWGG,GAAGNNNNNNTAC,GTANNNNNNCTTC,AACGGCNNNGTGC,GCACNNNGCCGTT,GCACGGCNNNGTT,AACNNNGCCGTGC,CGANNNNNNTGC,GCANNNNNNTCG,CGATCNNNNTGC,GCANNNNGATCG,TGANNNNNNTCA,TGATCNNNNTCA,TGANNNNGATCA,TGATCNNGATCA,AACCTGCNNGTGC,GCACNNGCAGGTT,GCACCTGCNNGTT,AACNNGCAGGTGC,GCCNNNNNGGC,AACNNNNNNGTGCMC,GKGCACNNNNNNGTT,CCWGGNCCWGG,AACTGGGNNGTGC,GCACNNCCCAGTT,GCACTGGGNNGTT,AACNNCCCAGTGC,GACNNNNGTC,GAGNNNNNCTC,CCWGGTCTC,GAGACCWGG,GATNNNNATC,GATCNNNATC,GATNNNGATC,GATCNNGATC,CCWGGCGCCWGG,ACNNNNNCTCC,GGAGNNNNNGT,AACNNNNNNGTGCAG,CTGCACNNNNNNGTT,GATCATGATC,CCWGGNNCCWGG,AACTGGNNNGTGC,GCACNNNCCAGTT,GCACTGGNNNGTT,AACNNNCCAGTGC,AACNAACCGGTGCGTGC,GCACGCACCGGTTNGTT,GCACNAACCGGTTNGTGC,AACGCACCGGTGCGTT,GCANNNNNTGC,GCACNNNNNNGTTCGAACNNNNNNGTGC,CCTNNNNNAGG,GCNNNNNNNGC,CCANNNNNNTGG,CCAGGNNCCTGG,CCTGGCCAGG,CAANNNNNGTGG,CCACNNNNNTTG,TTTAAACNNNNNNGTGC,GCACNNNNNNGTTTAAA,GCACNNNNNNGTTTAAACNNNNNNGTGC,CCAGGNCCY,RGGNCCTGG,CCWGGGNCCTGG,CCAGGNCCCWGG,AAGNNNNNCTT,GAYNNNNNVTC,GABNNNNNRTC,GAYNNNNNRTC,GATCNNNNVTC,GABNNNNGATC,GAYNNNNGATC,GATCNNNNRTC,GATCNNNGATC,GTYAACNNNNNNGTGC,GCACNNNNNNGTTRAC,GCACNNNNNNGTTAACNNNNNNGTGC,GATCNGATC,TAACTATAACGGTCCTAAGGTAGCGA,TCGCTACCTTAGGACCGTTATAGTTA,CTCTCTTAAGGTAGC,GCTACCTTAAGAGAG,TAGGGATAACAGGGTAAT,ATTACCCTGTTATCCCTA,GTTAACNNNNNNGTGC,GCACNNNNNNGTTAAC,AACNAACGCGTGCGTGC,GCACGCACGCGTTNGTT,GCACNAACGCGTTNGTGC,AACGCACGCGTGCGTT,TCCAACNNNNNNGTGC,GCACNNNNNNGTTGGA,CAYNNNNRTG,AACAYNNNNGTGC,GCACNNNNRTGTT,GTTTAAACNNNNNNGTGC,GCACNNNNNNGTTTAAAC,CCWGGCGCC,GGCGCCWGG,CCWGGNNCC,GGNNCCWGG,GATCGCGATC,AACNAACATGTGCGTGC,GCACGCACATGTTNGTT,GCACNAACATGTTNGTGC,AACGCACATGTGCGTT,TGGCAAACAGCTATTATGGGTATTATGGGT,ACCCATAATACCCATAATAGCTGTTTGCCA,ATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGG,CCATTTCATTACCTCTTTCTCCGCACCCGACATAGAT,AACNNGCACGTGCNNGTT,GAACNNNNNCTC,GAGNNNNNGTTC,CCAGGWCCY,RGGWCCTGG,CCWGGGWCCTGG,CCAGGWCCCWGG,CCAGGWCCTGG,TTATAACNNNNNNGTGC,GCACNNNNNNGTTATAA,GCACNNNNNNGTTATAACNNNNNNGTGC,GAACNNNNNNTAC,GTANNNNNNGTTC,GGCCNNNNNGGCC,AACNNACTAGTGC,GCACTAGTNNGTT,AACTAGTNNGTGC,GCACNNACTAGTT,AACNAACTAGTGCGTGC,GCACGCACTAGTTNGTT,GCACNAACTAGTTNGTGC,AACGCACTAGTGCGTT,AACNNNAACGTGCNNGTGC,GCACNNGCACGTTNNNGTT,TTAACNNNNNNGTGC,GCACNNNNNNGTTAA,CACNNNNNNTCC,GGANNNNNNGTG,CACNNNNGATCC,GGATCNNNNGTG,GATCTAGATC,CCANNNNNNNNNTGG,CCAGGCCT,AGGCCTGG,CCTCGAGG,GATCCGGA,TCCGGATC,GGCGCGCC,GCGATCGC,CCWGGWCC,GGWCCWGG,GATCGATC,GATCATGA,TCATGATC,GCGGCCGC,CCTGGCCR,YGGCCAGG,CCWGGNCC,GGNCCWGG,GGCCGGCC,RTGCGCAY,GATCNNGA,TCNNGATC,CGCGCGCG,CCTGGCCA,TGGCCAGG,CGCCGGCG,GTTTAAAC,GATCGCGA,TCGCGATC,TTAATTAA,VCTCGAGB,CCTGCAGG,CRCCGGYG,CGTCGACG,ATTTAAAT,GCCCGGGC,GATCTAGA,TCTAGATC,CACCTGC,GCAGGTG,CCTNAGG,GATCGAT,ATCGATC,CCTCAGC,GCTGAGG,GCTNAGC,CCTNAGC,GCTNAGG,GCTCTTC,GAAGAGC,GGTNACC,CGGWCCG,ACCWGGT,RGGNCCY,CCTGGAG,CTCCAGG,GGTGATC,GATCACC,GATCNGA,TCNGATC,GGGWCCC,GAAGATC,GATCTTC,CCCWGGG,TCCNGGA,TCCWGGA,RGGWCCY,TTATAA,AGGCCT,GACGTC,GTMKAC,TCCGGA,TGCGCA,ACCTGC,GCAGGT,GGTACC,AGTACT,GGYRCC,CCGCTC,GAGCGG,AACGTT,YGGCCR,RAATTY,CTGAAG,CTTCAG,CACGTG,GRCGYC,AGCGCT,CTTAAG,ACRYGT,ACCGGT,ACTAGT,CACGTC,GACGTG,GWGCWC,GTGCAC,CYCGRG,GGGCCC,ATTAAT,CCTAGG,TTCGAA,GCTAGC,GKGCMC,TGGCCA,GGATCC,GRGCYC,ATCGAT,CACGAG,CTCGTG,GGCGCC,GAAGAC,GTCTTC,GCATGC,GTATCC,GGATAC,TGATCA,ACTGGG,CCCAGT,CTRYAG,RGCGCY,AGATCT,GGNNCC,CTGGAG,CTCCAG,CTTGAG,CTCAAG,CGATCG,GGTCTC,GAGACC,YACGTR,CCNNGG,GAATGC,GCATTC,WCCGGW,GCAATG,CATTGC,RCCGGY,GCGCGC,GAGGAG,CTCCTC,CGGCCG,CCCAGC,GCTGGG,GTGCAG,CTGCAC,CGRYCG,CGTACG,CGTCTC,GAGACG,CCATGG,TCGCGA,GDGCHC,TGTACA,TCATGA,ACATGT,GTATAC,CCWWGG,CTCTTC,GAAGAG,GCNNGC,CCRYGG,GTTAAC,RCATGY,TACGTA,RGATCY,GCGATG,CATCGC,GCAGTG,CACTGC,CCCGGG,CCGCGG,TTTAAA,GGCGGA,TCCGCC,GAGCTC,GATATC,CTGRAG,CTYCAG,CAGCAG,CTGCTG,GAATTC,ATGCAT,CATATG,GTYRAC,AAGCTT,GTNNAC,TCNNGA,CAATTG,ACGCGT,TCCRAC,GTYGGA,GCCGGC,CMGCKG,GCCGAG,CTCGGC,CTCGAG,CTGCAG,CAGCTG,GTCGAC,CTYRAG,AATATT,GATCGA,TCGATC,GACCGA,TCGGTC,WGTACW,TARCCA,TGGYTA,TCTAGA,GGATC,GATCC,TTSAA,CCWGG,GTCTC,GAGAC,GCWGC,GGNCC,CCSGG,GGTGA,TCACC,GGWCC,GCAGC,GCTGC,CCATC,GATGG,ACGGC,GCCGT,GCNGC,CCNGG,GCATC,GATGC,ACTGG,CCAGT,GGATG,CATCC,CTCAG,CTGAG,GGGAC,GTCCC,ACNGT,CTNAG,CAGTG,CACTG,GACGC,GCGTC,CCCGC,GCGGG,GANTC,CGWCG,TCNGA,CCTTC,GAAGG,GCTCC,GGAGC,GTNAC,GAAGA,TCTTC,GAGTC,GACTC,GTSAC,GAWTC,GCSGC,CASTG,ATGAA,TTCAT,ACGGA,TCCGT,CGCG,CCGC,GCGG,GTAC,AGCT,GCGC,CTAG,GATC,GGCC,CCGG,CATG,RGCY,YATR,ACGT,TGCA,CCDG,CHGG,AATT,CCTC,GAGG,TTAA,ASST,TCGA                                                                                                                                                                                                                                                                                                                                                             5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               
         
                                       
   
      /                                                                                                                    ,                                                                                    -                                                                                     
   #   /                                 G   .   
                  
            #   !                           0                  $                  $         	            7   5   %   8   +      2   2   $   '      k               &   5   7   %      ,                                                                                                                                                               5                                                                                                                                                                                                                                                                                                 
   
             
 
  /                                        ,                              -                             
 # /           G . 
      
    # !         0      $      $   	    7 5 % 8 +  2 2 $ '  k     & 5 7 %  ,  4          "  j(/f=.%b;RO/fiZ["##z#)$$%3&8''	)&"8&f%lw. EC	
B)!J"%uAu \bC	}#77g%a&k <%O O   8\e<ERbC369NU^o,Obd"#d%%%G&P&[''(l'#r(5Ct!g"u#~&&Ct&&&Ct&g"	m!8&&g(VV#M%)v &q	#
o f%`&kT#o b!##C%v |M%#zg8 M%#%)v =!S zg[+G &6'$&& z  >
UO:#(($A%
: ]O:V	7 ""O:#!m"#P'1 #
h
&x k,H &7'a&) Y+Va
&W!#%)Yz#&% ,7
F	":|tg\v1!-'_d`igBG((A$s%&2(	y_%1  Y#	
ht[s!$(  (k6		((}h!!.#'w'	\`"m#('&S
3 ,	 }!T'(|g\v-'
 lkR(!&(Zz 	 YW%%u(DD;
IBEsyKf0%|;[SJ  Yg^ 0Ykv+VaCd



&aWVOP:;!###P%%%&((()%6_E"A%?\	#y&_{O3$-/ 7 8  g
E]`a/j #%)<U&x Ak  &a@  !S}9K-& )|)%%v&1tH;b'_{1!? E] l,k6		 R}((}h!!!.#&'T'w'(
 !C3
s!gE]/ # 
!&6V0:,7$8nNm@D^

CSZ!KTQ*@JNdQ}3 #d'  E]Ds!(/Uaa	r	
9 !A$:1'y!e$i#$` h{/	
WdNK* Mk(!%6_A$s%&2(E	y"A%_%(I
i Z!%%&_(ms
&qP#	
x&qvji  x  \DCF3
 k?k^>TyL+JS  "n##/$%z&$(!16U!'F  i	fM %($kZ\m#SJ fM %
&\\	?[m##y&
: 'W t (	 NV 
$'P)k !/"(8" Ua	
v  b2s	
1	: !g"F	

E
 g ^p{8kI*"'@(8 Vz?H$Y#j###C%M%%()0
P3"$ A
#goehRL  @!"#>$%%%%&a'((; {  d5VizX
BAb >NVxP; o  :!##P%%"((((()4S	`aW'	)	O1s ( [>	,OB-%p&&t({  5zXbxP;  :!#P%"((A
eL @!"#%%&( #gohR >$%%a'(l&vG#"%&0'Ja!z!%&( YLI0?'= @L O   <A\
X:
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Zk-֨جmk6Vm$hZ1lU6ZTf5flhEDlkK]0Ñ[iAMHƤØ*Te"jUѶƊm-[ ckrmK6ݜ-g+6VJU	YĀIsmKrs1T  <AdditionalSequenceProperties><UpstreamStickiness>0</UpstreamStickiness><DownstreamStickiness>0</DownstreamStickiness><UpstreamModification>Unmodified</UpstreamModification><DownstreamModification>Unmodified</DownstreamModification></AdditionalSequenceProperties>  W<AlignableSequences trimStringency="Medium"><Sequence name="MLM3636-dCas9-TET1-5.final" ID="0" use="0" sortOrder="0" trimmedRange="0..10514"/><Sequence name="MLM3636-dCas9-TET1-6.final" ID="1" sortOrder="1" trimmedRange="0..10514"/><Sequence name="MLM3636-dCas9-TET1-7.final" ID="2" sortOrder="2" trimmedRange="0..10513"/></AlignableSequences>  L      BC N sre```pp23*+/*IMbqqQjZjQj^r*& VU,6         BC =m1Rm8l[	d0l9px3`&@jw߽瞳vժU嫳/|vǏ__gǏȣ_Qz<eo<;JkmĒv'5gŖn߷vXmj塞f߹Bh!\<)t[kOʾ7KBLq*F<9nn|B71םVNjʺ'uB	k(czHFgR<y,ysэvη9m禔r)pO<_7q7.9XtG\/e<q!qpNۛwy焫ðSջlhݽ|nV\z{r]Uvk>q<HCLNg";N3Z
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  7<?xml version="1.0"?><Features nextValidID="19"><Feature recentID="0" name="AmpR promoter" directionality="2" type="promoter" swappedSegmentNumbering="1" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="2565-2669" color="#ffffff" type="standard"/><Q name="gene"><V text="&lt;html&gt;&lt;body&gt;&lt;i&gt;bla&lt;/i&gt;&lt;/body&gt;&lt;/html&gt;"/></Q></Feature><Feature recentID="1" name="AmpR" directionality="2" translationMW="31557.36" type="CDS" swappedSegmentNumbering="1" allowSegmentOverlaps="0" cleavageArrows="2495" readingFrame="-1" consecutiveTranslationNumbering="1" hitsStopCodon="1" detectionMode="exactProteinMatch"><Segment range="1704-2495" color="#ccffcc" type="standard" translated="1"/><Segment name="signal sequence" range="2496-2564" color="#ccffcc" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="gene"><V text="&lt;html&gt;&lt;body&gt;&lt;i&gt;bla&lt;/i&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="note"><V text="&lt;html&gt;&lt;body&gt;confers resistance to ampicillin, carbenicillin, and related antibiotics&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;β-lactamase&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"/></Q></Feature><Feature recentID="2" name="PuroR" directionality="1" translationMW="21496.48" type="CDS" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" hitsStopCodon="1"><Segment range="8-607" color="#ccffcc" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="gene"><V text="&lt;html&gt;&lt;body&gt;&lt;i&gt;pac&lt;/i&gt; from &lt;i&gt;Streptomyces alboniger&lt;/i&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="note"><V text="&lt;html&gt;&lt;body&gt;confers resistance to puromycin&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;puromycin &lt;i&gt;N&lt;/i&gt;-acetyltransferase&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"/></Q></Feature><Feature recentID="3" name="dCas9" directionality="1" translationMW="158331.48" type="CDS" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="4107-4133" color="#993366" type="standard" translated="1"/><Segment range="4134-4136" color="#cc99b2" type="standard" translated="1"/><Segment range="4137-6623" color="#993366" type="standard" translated="1"/><Segment range="6624-6626" color="#cc99b2" type="standard" translated="1"/><Segment range="6627-8210" color="#993366" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="locus_tag"><V text="&lt;html&gt;&lt;body&gt;&lt;br&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="note"><V text="&lt;html&gt;&lt;body&gt;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&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;catalytically dead mutant of the Cas9 endonuclease from the&amp;nbsp;&lt;i&gt;Streptococcus pyogenes&lt;/i&gt;&amp;nbsp;Type II CRISPR/Cas system&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="protein_id"><V text="&lt;html&gt;&lt;body&gt;&lt;br&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="MDKKYSIGL,A,IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVD,A,IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"/></Q></Feature><Feature recentID="4" name="gRNA scaffold" directionality="1" type="misc_RNA" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="3023-3098" color="#00ccff" type="standard"/><Q name="note"><V text="&lt;html&gt;&lt;body&gt;guide RNA scaffold for the &lt;i&gt;Streptococcus pyogenes&lt;/i&gt; CRISPR/Cas9 system&lt;/body&gt;&lt;/html&gt;"/></Q></Feature><Feature recentID="5" name="ori" directionality="2" type="rep_origin" swappedSegmentNumbering="1" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="945-1533" color="#ffff00" type="standard"/><Q name="direction"><V text="LEFT"/></Q><Q name="note"><V text="&lt;html&gt;&lt;body&gt;high-copy-number ColE1/pMB1/pBR322/pUC origin of replication&lt;/body&gt;&lt;/html&gt;"/></Q></Feature><Feature recentID="6" name="Kozak sequence" type="regulatory" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" originalName="Kozak sequence" originalSequence="GCCACCATGG" isFavorite="1"><Segment range="3930-3939" color="#a6acb3" type="standard"/><Q name="note"><V text="&lt;html&gt;&lt;body&gt;vertebrate consensus sequence for strong initiation of translation (&lt;a href=&quot;https://www.ncbi.nlm.nih.gov/pubmed/3313277&quot; title=&quot;https://www.ncbi.nlm.nih.gov/pubmed/3313277&quot;&gt;Kozak, 1987&lt;/a&gt;)&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="regulatory_class"><V predef="other"/></Q></Feature><Feature recentID="7" name="c-myc NLS" directionality="1" translationMW="997.21" type="CDS" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="3942-3968" color="#993366" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;nuclear localization signal of human c-Myc proto-oncogene&amp;nbsp;&lt;a href=&quot;http://www.ncbi.nlm.nih.gov/pubmed/3054508&quot; title=&quot;http://www.ncbi.nlm.nih.gov/pubmed/3054508&quot;&gt;(Dang and Lee, 1988)&lt;/a&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="PAAKRVKLD"/></Q></Feature><Feature recentID="8" name="c-myc NLS" directionality="1" translationMW="997.21" type="CDS" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="3975-4001" color="#993366" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;nuclear localization signal of human c-Myc proto-oncogene&amp;nbsp;&lt;a href=&quot;http://www.ncbi.nlm.nih.gov/pubmed/3054508&quot; title=&quot;http://www.ncbi.nlm.nih.gov/pubmed/3054508&quot;&gt;(Dang and Lee, 1988)&lt;/a&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="PAAKRVKLD"/></Q></Feature><Feature recentID="9" name="c-myc NLS" directionality="1" translationMW="997.21" type="CDS" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="10431-10457" color="#993366" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;nuclear localization signal of human c-Myc proto-oncogene&amp;nbsp;&lt;a href=&quot;http://www.ncbi.nlm.nih.gov/pubmed/3054508&quot; title=&quot;http://www.ncbi.nlm.nih.gov/pubmed/3054508&quot;&gt;(Dang and Lee, 1988)&lt;/a&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="PAAKRVKLD"/></Q></Feature><Feature recentID="10" name="CMV enhancer" geneticCode="-1" type="enhancer" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="3117-3402" color="#ffffff" type="standard"/><Q name="note"><V text="&lt;html&gt;&lt;body&gt;human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer&lt;/body&gt;&lt;/html&gt;"/></Q></Feature><Feature recentID="11" name="chicken β-actin promoter" directionality="1" type="promoter" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="3404-3681" color="#ffffff" type="standard"/></Feature><Feature recentID="12" name="SV40 NLS" directionality="1" translationMW="883.15" type="CDS" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" consecutiveNumberingStartsFrom="126" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="4077-4097" color="#993366" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="locus_tag"><V text="&lt;html&gt;&lt;body&gt;&lt;br&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="note"><V text="&lt;html&gt;&lt;body&gt;&lt;br&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;nuclear localization signal of SV40 (simian virus 40) large T antigen&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="protein_id"><V text="&lt;html&gt;&lt;body&gt;&lt;br&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="PKKKRKV"/></Q></Feature><Feature recentID="13" name="hybrid intron" type="intron" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="3682-3910" color="noColor" type="standard"/><Q name="note"><V text="&lt;html&gt;&lt;body&gt;hybrid between chicken β-actin (CBA) and minute virus of mice (MMV) introns (&lt;a href=&quot;http://www.ncbi.nlm.nih.gov/pubmed/21476867&quot; title=&quot;http://www.ncbi.nlm.nih.gov/pubmed/21476867&quot;&gt;Gray et al., 2011&lt;/a&gt;)&lt;/body&gt;&lt;/html&gt;"/></Q></Feature><Feature recentID="14" name="bGH poly(A) signal" type="polyA_signal" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="641-848" color="#a6acb3" type="standard"/><Q name="note"><V text="&lt;html&gt;&lt;body&gt;bovine growth hormone polyadenylation signal&lt;/body&gt;&lt;/html&gt;"/></Q></Feature><Feature recentID="15" name="U6 promoter" directionality="1" type="promoter" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="2755-2995" color="#ffffff" type="standard"/><Q name="note"><V text="&lt;html&gt;&lt;body&gt;RNA polymerase III promoter for human U6 snRNA&lt;/body&gt;&lt;/html&gt;"/></Q></Feature><Feature recentID="16" name="3xFLAG" directionality="1" translationMW="2730.71" type="CDS" allowSegmentOverlaps="0" cleavageArrows="4070" consecutiveTranslationNumbering="1" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="4005-4070" color="#cc99b2" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;three tandem FLAG® epitope tags, followed by an enterokinase cleavage site&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="DYKDHDGDYKDHDIDYKDDDDK"/></Q></Feature><Feature recentID="17" name="T2A" directionality="1" translationMW="1829.96" type="CDS" allowSegmentOverlaps="0" cleavageArrows="10511" consecutiveTranslationNumbering="1" consecutiveNumberingStartsFrom="139" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="10461-10514" color="#cc99b2" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="note"><V text="&lt;html&gt;&lt;body&gt;Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides.&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="product"><V text="&lt;html&gt;&lt;body&gt;2A peptide from&amp;nbsp;&lt;i&gt;Thosea asigna&lt;/i&gt; virus capsid protein&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="protein_id"><V text="&lt;html&gt;&lt;body&gt;&lt;br&gt;&lt;/body&gt;&lt;/html&gt;"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="EGRGSLLTCGDVEENPGP"/></Q></Feature><Feature recentID="18" name="TET1 catalytic domain" directionality="1" type="CDS" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" maxRunOn="607" maxFusedRunOn="607"><Segment range="8268-10412" color="#3366ff" type="standard" translated="1"/><Q name="codon_start"><V int="1"/></Q><Q name="transl_table"><V int="1"/></Q><Q name="translation"><V text="CSCLDRVIQKDKGPYYTHLGAGPSVAAVREIMENRYGQKGNAIRIEIVVYTGKEGKSSHGCPIAKWVLRRSSDEEKVLCLVRQRTGHHCPTAVMVVLIMVWDGIPLPMADRLYTELTENLKSYNGHPTDRRCTLNENRTCTCQGIDPETCGASFSFGCSWSMYFNGCKFGRSPSPRRFRIDPSSPLHEKNLEDNLQSLATRLAPIYKQYAPVAYQNQVEYENVARECRLGSKEGRPFSGVTACLDFCAHPHRDIHNMNNGSTVVCTLTREDNRSLGVIPQDEQLHVLPLYKLSDTDEFGSKEGMEAKIKSGAIEVLAPRRKKRTCFTQPVPRSGKKRAAMMTEVLAHKIRAVEKKPIPRIKRKNNSTTTNNSKPSSLPTLGSNTETVQPEVKSETEPHFILKSSDNTKTYSLMPSAPHPVKEASPGFSWSPKTASATPAPLKNDATASCGFSERSSTPHCTMPSGRLSGANAAAADGPGISQLGEVAPLPTLSAPVMEPLINSEPSTGVTEPLTPHQPNHQPSFLTSPQDLASSPMEEDEQHSEADEPPSDEPLSDDPLSPAEEKLPHIDEYWSDSEHIFLDANIGGVAIAPAHGSVLIECARRELHATTPVEHPNRNHPTRLSLVFYQHKNLNKPQHGFELNKIKFEAKEAKNKKMKASEQKDQAANEGPEQSSEVNELNQIPSHKALTLTHDNVVTVSPYALTHVAGPYNHWV"/></Q></Feature></Features>
  
<?xml version="1.0"?><Primers nextValidID="3"><HybridizationParams minContinuousMatchLen="10" allowMismatch="1" minMeltingTemperature="50" showAdditionalFivePrimeMatches="0" minimumFivePrimeAnnealing="25"/><Primer recentID="0" name="Gb_3636-dCas9-TET1-F" sequence="tcactttttttcaggttggataccctcgtaaaggccacc" description="&lt;html&gt;&lt;body&gt;&lt;/body&gt;&lt;/html&gt;" dateAdded="2023-07-06T17:59:58Z"><BindingSite location="3896-3934" boundStrand="0" annealedBases="tcactttttttcaggttggataccctcgtaaaggccacc" meltingTemperature="68"><Component hybridizedRange="3896-3934" bases="tcactttttttcaggttggataccctcgtaaaggccacc"/></BindingSite><BindingSite location="8516-8528" boundStrand="0" annealedBases="cgtaaggccacc" meltingTemperature="39"><Component bases="tcactttttttcaggttggataccct"/><Component hybridizedRange="8516-8519" bases="cgta"/><Component bases="a"/><Component hybridizedRange="8521-8528" bases="aggccacc"/></BindingSite><BindingSite simplified="1" location="3896-3934" boundStrand="0" annealedBases="tcactttttttcaggttggataccctcgtaaaggccacc" meltingTemperature="68"><Component hybridizedRange="3896-3934" bases="tcactttttttcaggttggataccctcgtaaaggccacc"/></BindingSite><BindingSite simplified="1" location="8516-8528" boundStrand="0" annealedBases="cgtaaggccacc" meltingTemperature="39"><Component bases="tcactttttttcaggttggataccct"/><Component hybridizedRange="8516-8519" bases="cgta"/><Component bases="a"/><Component hybridizedRange="8521-8528" bases="aggccacc"/></BindingSite></Primer><Primer recentID="1" name="Gb_3636-dCas9-TET1-R" sequence="tgtactcggtcatggtggcaccagggccgggattctcctc" description="&lt;html&gt;&lt;body&gt;&lt;/body&gt;&lt;/html&gt;" dateAdded="2023-07-06T18:00:40Z"><BindingSite location="10496-19" boundStrand="1" annealedBases="tgtactcggtcatggtggcaccagggccgggattctcctc" meltingTemperature="75"><Component hybridizedRange="10496-19" bases="tgtactcggtcatggtggcaccagggccgggattctcctc"/></BindingSite><BindingSite simplified="1" location="10496-19" boundStrand="1" annealedBases="tgtactcggtcatggtggcaccagggccgggattctcctc" meltingTemperature="75"><Component hybridizedRange="10496-19" bases="tgtactcggtcatggtggcaccagggccgggattctcctc"/></BindingSite></Primer><Primer recentID="2" name="MLM3636_727F" sequence="aagcaggctttaaaggaacca" description="&lt;html&gt;&lt;body&gt;&lt;/body&gt;&lt;/html&gt;" dateAdded="2024-03-20T15:53:50Z"><BindingSite location="2715-2735" boundStrand="0" annealedBases="aagcaggctttaaaggaacca" meltingTemperature="56"><Component hybridizedRange="2715-2735" bases="aagcaggctttaaaggaacca"/></BindingSite><BindingSite simplified="1" location="2715-2735" boundStrand="0" annealedBases="aagcaggctttaaaggaacca" meltingTemperature="56"><Component hybridizedRange="2715-2735" bases="aagcaggctttaaaggaacca"/></BindingSite></Primer></Primers>
  '<Notes>
<UUID>e1c7f9f9-e803-4141-a6fd-25bf07e3ec38</UUID>
<Type>Synthetic</Type>
<ConfirmedExperimentally>0</ConfirmedExperimentally>
<Description>&lt;html>&lt;body>Empty vector for transient transfection of dCas9-TET1. Clone gRNA using BsmBI digestion.&lt;/body>&lt;/html></Description>
<Created UTC="17:32:1">2023.7.6</Created>
<LastModified UTC="15:53:50">2024.3.20</LastModified>
<SequenceClass>UNA</SequenceClass>
<TransformedInto>unspecified</TransformedInto>
<Comments>&lt;html>&lt;body>Digest MLM3636-Puro with AgeI.&lt;br/>Single 6kb PCR fragment from CLYBL-dCas9-TET1 for Gibson Assembly.&lt;br/>Possible requirement for gel purification of the dCas9-TET1 fragment to avoid complications during PCR from the other T2A sequences present in CLYBL-dCas9-TET1?&lt;/body>&lt;/html></Comments>
</Notes>
  Y     K         Unsaved Enzyme Set                                                                                           d     THO              E              Y? P                                                                                                                                                              3<?xml version="1.0"?><EnzymeVisibilities vals=""/>
  <?xml version="1.0"?><CustomEnzymeSets><CustomEnzymeSet type="2" name="Unsaved Enzyme Set" enzymeNames="AleI ApaI AscI BbvCI BsiWI BsmBI BspDI BstEII BstXI ClaI CsiI FspAI MfeI NaeI NgoMIV NruI PmlI PshAI PspOMI PspXI SbfI SexAI SmaI SnaBI SphI TspMI XbaI XmaI"/></CustomEnzymeSets>
