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vvz"& ^ c@G-	g	
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X 	K	d			
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UfQ
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E-N3BNo{/Ezez
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I#c$$$%'')i)6  7 f77		
cLvU9 nIT%5t  ""#P%%&f&N''(() G 6X70A[tR!$"8""l#A&j&&&$'3''((n)D 6 ` 56t
56JKST78sH4"#b$$$O%%&e&&#'1'2'M''((  :  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                                                                                                                                                                                                                                                                                                                                                             9                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
         
                                          
      0                                                                                                                   	   -                                                                              	      -                                                                                     	   #   /                                 L   .   
                  
            #   "                           /                  %                  )         	             7   ;   &   9   -      2   2   &   '      k                '   5   7   &      ,                                                                                                                                                               9                                                                                                                                                                                                                                                                                                
   
              
  0                                         	 -                            	  -                             	 # /           L . 
      
    # "         /      %      )   	     7 ; & 9 -  2 2 & '  k      ' 5 7 &  ,  T    H      r  2(=%b;R
ca{,#l#m##$%%&'(i)/'N#{&f1&. C	
!
"o&p5  \bR	e#$@%&k
!<0&a{ O   8\e<ERbj+R
&^p##%*&0&&&'C()HI' W$#5Z2""#&/''/'Y'/'"	!&P'(VV[$%o)v &q	 W$jo %&k#o b!-$Q$%v %  X$"zl8 %  X$?&l)v "^"_ z[\ 9''/[%>'Hx>' z { >
lX W$((%C@y ]PV	7 R]" ]" l W$#$P_(1 #
>
x [ :''/a
ttx Y+Va
  "^$'&r)Y#% ,7
o!F	":|tn!'_PW4X()T8%%'(	%M1  Y#	
>' !1hsO"t$(~ A!6Z)k6		<!"#k''	\IC\AI##(u&)
 ,	`E"'(|'
? lkE "~&(+ 	 Y	%&I)DgD;
sK+6c I%|;q]  Yg!1d 0Ykv+VaCd



 Hl? ")$M$^$%'&\&}&(()r)E#%\	#&_3+%BM 7 8  g
cFRS^ -$A&n) 'x A
~Q~ A!&a@~ A!!$&tA'`)5%^&&H
P'_!?  l,k6		`<EE !""#~&k'''(
<!L"C3
$sO"g
^ -$ 
!&6
T$8nNm@D^

dR$&t#@R ~#'  cDsO"Q)Uaa	r	
Rd?x 
$'y~!$z#% ch{/	
ly[S Mk"T8%%'(E	#%%)j !4&7&<'_X)m&G
qP7|(%n$	
(q:L3 ;!=!x  \DCF3
$_g	,S[m@	 `"##$&&(c!1-!'y  i	c5 1&)$k\A#q]  1&
\\	hA##&
' t 	 DV 
9ftvtJx<!L""(v"Ua	
RA?v  b2s	
@yi ^""F	

E
z.H,")((8 Vz?W+lFS^# #[$%%B&r(o)0
3E8Ad"+% A
#d.  ![##$%0&3&6&&'v(!); {  d5VizX
Q.H`mYZR   |   !N$X$%?&((())l)4S	:'i)	2l W) [>	CE%&8'({  5zXm   |  !X$%()A
d. ![##%0&&v( # $3&6&'!)@6Q$%''JbEtqW!!<&z&6) Y,5)'= L O   <A\
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VPP:aȌ&9m2NXӭۜh-p֦m0fRZ,sˀLu9s3̹AAI&8VƪX+䪴MdֶI[MFd(6XsR؊E*R4F	m6͹&،H[A9iUrj(2	l9"2'ٚͩKX,`1&t4lH͍(#)6`d+sR@1+"XrL&4HM3[[KʨFd,t4a 4pI9WkN ʑ-fLAes"irS)"YstmYEBKIG  <AdditionalSequenceProperties><UpstreamStickiness>0</UpstreamStickiness><DownstreamStickiness>0</DownstreamStickiness><UpstreamModification>Unmodified</UpstreamModification><DownstreamModification>Unmodified</DownstreamModification></AdditionalSequenceProperties>  c<AlignableSequences trimStringency="Medium"><Sequence name="MLM3636-dCas9-TET1-CGG-1.final" ID="0" sortOrder="0" trimmedRange="0..10609"/><Sequence name="MLM3636-dCas9-TET1-CGG-2.final" ID="1" sortOrder="1" trimmedRange="0..10608"/><Sequence name="MLM3636-dCas9-TET1-CGG-3.final" ID="2" use="0" sortOrder="2" trimmedRange="0..10609"/></AlignableSequences>        BC N sre```pp23*+/*IMbqqQjZjQj^r*C& s6         BC OuQC҈-7Cܨ 写L 冇@zCb ֹt@~s>{UV~}7g_?_|׿ꇯo_=C逸5y>;zS%-^j,ˋ>V{0Ҭ[,y:!-Y{mrl:N׿4)WzJ?8ipN\˺9hg5yf;䡕FN!5gmĥ8cayW
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  8<?xml version="1.0"?><Features nextValidID="20"><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="4203-4229" color="#993366" type="standard" translated="1"/><Segment range="4230-4232" color="#cc99b2" type="standard" translated="1"/><Segment range="4233-6719" color="#993366" type="standard" translated="1"/><Segment range="6720-6722" color="#cc99b2" type="standard" translated="1"/><Segment range="6723-8306" 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="3045-3120" 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="4026-4035" 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="4038-4064" 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="4071-4097" 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="10527-10553" 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="3213-3498" 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="3500-3777" 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="4173-4193" 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="3778-4006" 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="2776-3016" 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="4166" consecutiveTranslationNumbering="1" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="4101-4166" 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="10607" consecutiveTranslationNumbering="1" consecutiveNumberingStartsFrom="139" maxRunOn="607" maxFusedRunOn="607" detectionMode="exactProteinMatch"><Segment range="10557-10610" 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" translationMW="78821.01" type="CDS" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1" maxRunOn="607" maxFusedRunOn="607"><Segment range="8364-10508" 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><Feature recentID="19" name="CGG6" type="misc_feature" allowSegmentOverlaps="0" consecutiveTranslationNumbering="1"><Segment range="3025-3044" color="#ff0000" type="standard"/></Feature></Features>
  <?xml version="1.0"?><Primers nextValidID="2"><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="3992-4030" boundStrand="0" annealedBases="tcactttttttcaggttggataccctcgtaaaggccacc" meltingTemperature="68"><Component hybridizedRange="3992-4030" bases="tcactttttttcaggttggataccctcgtaaaggccacc"/></BindingSite><BindingSite location="8612-8624" boundStrand="0" annealedBases="cgtaaggccacc" meltingTemperature="39"><Component bases="tcactttttttcaggttggataccct"/><Component hybridizedRange="8612-8615" bases="cgta"/><Component bases="a"/><Component hybridizedRange="8617-8624" bases="aggccacc"/></BindingSite><BindingSite simplified="1" location="3992-4030" boundStrand="0" annealedBases="tcactttttttcaggttggataccctcgtaaaggccacc" meltingTemperature="68"><Component hybridizedRange="3992-4030" bases="tcactttttttcaggttggataccctcgtaaaggccacc"/></BindingSite><BindingSite simplified="1" location="8612-8624" boundStrand="0" annealedBases="cgtaaggccacc" meltingTemperature="39"><Component bases="tcactttttttcaggttggataccct"/><Component hybridizedRange="8612-8615" bases="cgta"/><Component bases="a"/><Component hybridizedRange="8617-8624" 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="10592-19" boundStrand="1" annealedBases="tgtactcggtcatggtggcaccagggccgggattctcctc" meltingTemperature="75"><Component hybridizedRange="10592-19" bases="tgtactcggtcatggtggcaccagggccgggattctcctc"/></BindingSite><BindingSite simplified="1" location="10592-19" boundStrand="1" annealedBases="tgtactcggtcatggtggcaccagggccgggattctcctc" meltingTemperature="75"><Component hybridizedRange="10592-19" bases="tgtactcggtcatggtggcaccagggccgggattctcctc"/></BindingSite></Primer></Primers>
  '<Notes>
<UUID>7766a762-df03-48d8-991b-bbc37a0c5808</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="15:18:42">2023.7.26</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 BspDI BstEII BstXI ClaI CsiI FspAI MfeI NaeI NgoMIV NruI PmlI PshAI PspOMI PspXI SbfI SexAI SmaI SnaBI SphI TspMI XbaI XmaI"/></CustomEnzymeSets>
