LOCUS       sequence-403324-       17970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive CRISPRi by Zim3-dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17970)
  TITLE     CRISPRi by Zim3-dCas9 for iPSC Neurodegenerative Disease Initiative 
            (iNDI)
REFERENCE   2  (bases 1 to 17970)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: iE61 PB-Zim3-XTEN-dCas9-mScarlet-puro-BFP
FEATURES             Location/Qualifiers
     source          1..17970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        9..388
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..666
                     /label=chicken beta-actin promoter
     intron          667..1683
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1607..1629
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1691..1710
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(2158..2184,2185..2187,2188..4674,4675..4677,
                     4678..6261)
                     /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             6271..6297
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6316..6336
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6343..6363
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6379..6432
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6433..6459
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6475..6501
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     misc_feature    7249..7837
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7302..7322)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7720..7731)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7869..8345
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(8068..8087)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   8790..8852
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8982..8999)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8982..8998)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8991..9013)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9140..9595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9227..9246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9437..9458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9621..9725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9726..9794,9795..10586)
                     /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     complement(9944..9963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10757..11345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11246..11265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11499..11516
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11633..11654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11669..11674,11675..11692,11693..11699)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11707..11723
                     /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)."
     primer_bind     11712..11734
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11731..11747
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11731..11747
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   12238..12272
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    12788..12821
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    12822..13046
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(13066..13776)
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     regulatory      13773..13782
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(13804..13830)
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             complement(13846..13872)
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             complement(13879..13932)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(13942..14538)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(14022..14042)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     14519..14538
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     intron          14561..15499
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(14584..14604)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(15500..15711)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    15712..15745
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ggcgcgccga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
       61 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      121 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      181 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      241 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      301 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      361 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggg sgcgcgccag gcggggcggg gcggggcgag
      541 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
      601 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      661 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      721 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      781 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
      841 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
      901 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
      961 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     1021 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     1081 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     1141 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     1201 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     1261 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     1321 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     1381 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     1441 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     1501 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     1561 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     1621 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     1681 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaaggtacc
     1741 tcgcgagccg ccaccatgaa caactctcag ggcagagtga ccttcgagga cgtgaccgtg
     1801 aacttcaccc aaggcgagtg gcagagactg aaccccgagc agagaaacct gtaccgggac
     1861 gtgatgctgg aaaactacag caacctggtg tccgtcggcc agggcgagac aacaaagcct
     1921 gacgtgatcc tgcggctgga acagggcaaa gagccttggc tggaagagga agaggtgctc
     1981 ggatctggca gagccgagaa gaatggcgac atcggcggac agatctggaa gcccaaggac
     2041 gtgaaagagt ctctgagtgg cggctcaagt ggcgggtcta gtgggagtga aacgccaggg
     2101 acttcagaat cagcaacccc ggaatcatcc ggcggatcat ccggtgggtc tgtatacatg
     2161 gacaagaagt actctatcgg cctggccatc ggcaccaatt ctgttggatg ggccgtgatc
     2221 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     2281 agcatcaaga agaatctgat cggcgccctg ctgttcgact ctggcgaaac agccgaagcc
     2341 accagactga agagaaccgc cagacggcgg tacaccagaa gaaagaaccg gatctgctac
     2401 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     2461 gaagagtcct tcctggtcga ggaagataag aagcacgagc ggcaccccat cttcggcaac
     2521 atcgtggatg aggtggccta ccacgagaag taccctacca tctaccacct ccggaagaaa
     2581 ctggtggaca gcaccgacaa ggccgacctg agactgatct atctggccct ggctcacatg
     2641 atcaagttcc ggggccactt tctgatcgag ggcgacctga atcctgacaa cagcgacgtg
     2701 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     2761 aacgccagcg gagtggatgc caaggccatc ctgtctgcca gactgagcaa gagcagacgc
     2821 ctggaaaacc tgatcgctca gctgcccggc gagaagaaga acggcctgtt cggaaacctg
     2881 attgccctga gcctgggcct gacacctaac ttcaagagca acttcgacct ggctgaggac
     2941 gccaaactgc agctgtccaa ggacacctac gacgacgacc tggacaatct gctggcccag
     3001 atcggcgatc agtacgccga cttgtttctg gccgccaaga acctgtccga cgccatcctg
     3061 ctgagcgaca tcctgagagt gaacaccgag atcacaaagg cccctctgag cgcctctatg
     3121 atcaagagat acgacgagca ccaccaggat ctgaccctgc tgaaggccct cgttagacag
     3181 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     3241 tacattgatg gcggagccag ccaagaggaa ttttacaagt tcatcaagcc catccttgag
     3301 aagatggacg gcaccgagga actgctggtc aagctgaaca gagaggacct gctgcggaag
     3361 cagcggacct tcgacaatgg ctctatccct caccagatcc acctgggaga gctgcacgcc
     3421 attctgcgga gacaagagga cttttaccca ttcctgaagg acaaccggga aaagattgag
     3481 aagatcctga ccttcagaat cccctactac gtgggaccac tggccagagg caatagcaga
     3541 ttcgcctgga tgaccagaaa gagcgaggaa accatcacac cctggaactt cgaggaagtg
     3601 gtggacaagg gcgccagcgc tcagtccttc atcgagcgga tgaccaactt cgataagaac
     3661 ctgcctaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     3721 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc ctttctgagc
     3781 ggcgagcaga aaaaggccat tgtggatctg ctgttcaaga ccaaccggaa agtgacagtg
     3841 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgacagcgt ggaaatcagc
     3901 ggcgtggaag atcggttcaa tgccagcctg ggcacatacc acgacctgct gaaaattatc
     3961 aaggacaagg acttcctgga caacgaagag aacgaggaca tcctggaaga catcgtgctg
     4021 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac atacgcccac
     4081 ctgttcgacg acaaagtgat gaagcaactg aagcggcgga gatacaccgg ctggggtaga
     4141 ctgtcccgga agctgatcaa cggcatccgg gataagcagt ccggcaagac catcctggac
     4201 tttctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     4261 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccaaggcga ttctctgcac
     4321 gagcacattg ccaacctggc cggatctccc gccattaaga agggcatcct gcagaccgtg
     4381 aaggtggtgg acgagcttgt gaaagtgatg ggcagacaca agcccgagaa catcgtgatc
     4441 gaaatggcca gagagaacca gaccacacag aagggccaga agaacagccg cgagagaatg
     4501 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     4561 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tggacgggat
     4621 atgtacgtgg accaagagct ggacatcaac cggctgtccg actacgatgt ggacgctatc
     4681 gtgccccagt cttttctgaa ggacgactcc atcgacaaca aggtcctgac cagaagcgac
     4741 aagaaccggg gcaagagcga taacgtgccc tccgaagagg tggtcaagaa gatgaagaac
     4801 tattggcgcc agctgctgaa tgccaagctg attacccagc ggaagttcga caacctgacc
     4861 aaggccgaaa gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     4921 gtggaaaccc ggcagatcac caaacacgtg gcacagattc tggactcccg gatgaacact
     4981 aagtacgacg agaacgacaa actgatccgg gaagtgaaag tcatcaccct gaagtccaag
     5041 ctggtgtctg atttccggaa ggatttccag ttctacaaag tgcgcgagat caacaactac
     5101 catcacgccc acgacgccta cctgaatgcc gttgttggaa cagccctgat caagaagtat
     5161 cccaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     5221 atcgccaaga gcgagcaaga gattggcaag gctaccgcca agtacttttt ctactccaac
     5281 atcatgaact ttttcaagac agagatcacc ctggccaacg gcgagatccg gaaaagaccc
     5341 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggcag agattttgcc
     5401 acagtgcgga aagtgctgag catgccccaa gtgaatatcg tgaagaaaac cgaggtgcag
     5461 acaggcggct tcagcaaaga gtctatcctg cctaagcgga acagcgacaa gctgatcgcc
     5521 cggaagaagg actgggaccc aaagaagtac ggcggcttcg atagccctac cgtggcctat
     5581 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactcaa gagcgtgaaa
     5641 gaactgctcg gcatcaccat catggaacgg tccagctttg agaagaaccc tattgatttc
     5701 cttgaggcca agggctacaa agaagtgaaa aaggacctga tcatcaagct ccccaagtac
     5761 tccctgttcg agctggaaaa tggccggaag cggatgctgg cttctgctgg cgaactgcag
     5821 aagggaaacg aactggccct gcctagcaaa tatgtgaact ttctgtacct ggccagccac
     5881 tatgagaagc tgaagggcag ccccgaggac aatgagcaaa agcagctgtt tgtcgagcag
     5941 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     6001 ctggctgacg ccaatctgga caaagtgctg tccgcctaca acaagcaccg ggacaagcct
     6061 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaacct gggagcccct
     6121 gccgccttca agtacttcga caccaccatc gaccggaagc gctacaccag caccaaagag
     6181 gttctggacg ccacactgat ccaccagagc atcaccggcc tgtacgagac aagaatcgac
     6241 ctgtctcagc tcggcggcga tcctcgaggc tatccctatg acgtgcccga ttatgccagc
     6301 agtggcagcg gctcccccaa gaaaaaacgc aaggtggaag atcctaagaa aaagcggaaa
     6361 gtggttaacg gaagtggcga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     6421 aatcctggcc cacctgccgc taaacgggtc aaactcgacg gcggttctgg aggtcctgct
     6481 gcaaagcgcg ttaagttgga tgctagcatg gaatttgtgt ctaaagggga agcagtcatt
     6541 aaagagttca tgcgcttcaa agtacacatg gaaggaagta tgaacgggca cgaatttgaa
     6601 atagaaggcg aaggggaagg tcggccatac gaagggacgc aaaccgcgaa gttgaaggtt
     6661 acaaagggag gaccgttgcc ctttagctgg gacatcctgt ctccacagtt tatgtacggt
     6721 tcaagagctt ttactaagca ccccgctgac atcccggact actataaaca gtcattcccc
     6781 gaaggattca aatgggagag ggtcatgaat ttcgaggacg gaggtgctgt tacagttacg
     6841 caggatacct cacttgagga tggcacgctt atatataagg tgaagctccg cggtacgaac
     6901 tttccgccgg acggccccgt tatgcagaag aagacgatgg gctgggaagc ctctacggaa
     6961 cgcctgtatc ctgaggacgg tgttctgaaa ggggatatta aaatggccct tcggttgaaa
     7021 gatgggggcc gctatcttgc ggactttaag accacctaca aagcaaaaaa gcctgtccaa
     7081 atgcccggtg catataacgt cgatagaaaa cttgatataa cctcccataa tgaagattac
     7141 acagtagtcg agcagtacga acgaagcgaa ggtcggcact caactggagg tatggatgaa
     7201 ttgtataagt aggatatcat gcctaatttt gagtgtatta ctttcgaaaa tcaacctctg
     7261 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     7321 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     7381 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     7441 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     7501 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     7561 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     7621 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc
     7681 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     7741 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     7801 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc gctggtcgac
     7861 agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg
     7921 ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact
     7981 aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg
     8041 ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac
     8101 aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc
     8161 ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt
     8221 agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct
     8281 acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg
     8341 tccttgttta aacgcgatcg cttaattaag gccgtggagg ccttttcccc gtatcccccc
     8401 aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat cccgtgccac
     8461 cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg gggagcgccg
     8521 gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg gacgtaatta
     8581 catccctggg ggctttgggg gggggctgtc cctcataaaa gttttgttac tttatagaag
     8641 aaattttgag tttttgtttt ttttaataaa taaataaaca taaataaatt gtttgttgaa
     8701 tttattatta gtatgtaagt gtaaatataa taaaacttaa tatctattca aattaataaa
     8761 taaacctcga tatacagacc gataaaacac atgcgtcaat tttacacatg attatcttta
     8821 acgtacgtca caatatgatt atctttctag ggttaatcta gctgcgtgtt ctgcagcgtg
     8881 tcgagcatct tcatctgctc catcacgctg taaaacacat ttgcaccgcg agtctgcccg
     8941 tcctccacgg gttcaaaaac gtgaatgaac gaggcgcgct cactggccgt cgttttacaa
     9001 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     9061 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     9121 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     9181 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     9241 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     9301 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     9361 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     9421 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     9481 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     9541 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     9601 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     9661 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     9721 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     9781 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     9841 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     9901 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     9961 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
    10021 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
    10081 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
    10141 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
    10201 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
    10261 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
    10321 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
    10381 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
    10441 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
    10501 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
    10561 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
    10621 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
    10681 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
    10741 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
    10801 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
    10861 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
    10921 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
    10981 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
    11041 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
    11101 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
    11161 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
    11221 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
    11281 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
    11341 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
    11401 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
    11461 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
    11521 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
    11581 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
    11641 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
    11701 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
    11761 agcgcgcccg ccgggtaact cacggggtat ccatgtccat ttctgcggca tccagccagg
    11821 atacccgtcc tcgctgacgt aatatcccag cgccgcaccg ctgtcattaa tctgcacacc
    11881 ggcacggcag ttccggctgt cgccggtatt gttcgggttg ctgatgcgct tcgggctgac
    11941 catccggaac tgtgtccgga aaagccgcga cgaactggta tcccaggtgg cctgaacgaa
    12001 cagttcaccg ttaaaggcgt gcatggccac accttcccga atcatcatgg taaacgtgcg
    12061 ttttcgctca acgtcaatgc agcagcagtc atcctcggca aactctttcc atgccgcttc
    12121 aacctcgcgg gaaaaggcac gggcttcttc ctccccgatg cccagatagc gccagcttgg
    12181 gcgatgactg agccggaaaa aagacccgac gatatgatcc tgatgcagct agattaaccc
    12241 tagaaagata gtctgcgtaa aattgacgca tgcattcttg aaatattgct ctctctttct
    12301 aaatagcgcg aatccgtcgc tgtgcattta ggacatctca gtcgccgctt ggagctcccg
    12361 tgaggcgtgc ttgtcaatgc ggtaagtgtc actgattttg aactataacg accgcgtgag
    12421 tcaaaatgac gcatgattat cttttacgtg acttttaaga tttaactcat acgataatta
    12481 tattgttatt tcatgttcta cttacgtgat aacttattat atatatattt tcttgttata
    12541 gaatcaggga cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta
    12601 gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc cgcatccccg
    12661 agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat cgctttcctc
    12721 tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg ggaaaaagcc
    12781 ataccaaata acttcgtata atgtatgcta tacgaagtta tccatagagc ccaccgcatc
    12841 cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc
    12901 ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga
    12961 aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac
    13021 agatggctgg caactagaag gcacagaatt cttagcggcc gcctaattaa gcttgtgccc
    13081 cagtttgcta gggaggtcgc agtatctggc cactgccacc tcgtgctgct cgacgtaggt
    13141 ctcgttgttg gcctccttga ttctttccag tctgtagtcc acatagtaga cgccaggcat
    13201 cttgaggttc ttagcgggtt tcttggatct atatgtggtc ttggcgtttg cgatcagatg
    13261 gctcccgccc acgagcttca gggccatgtc gtttctgcct tccaggccgc cgtcagcggg
    13321 gtacagcgtc tcggtgaagg cctcccagcc gagtgttttc ttctgcatca cagggccgtt
    13381 ggatgtgaag ttcacccctc tgatcttgac gttgtagatg aggcagccgt cctggaggct
    13441 ggtgtcctgg gtagcggtca gcacgccccc gtcttcgtat gtggtgactc tctcccatgt
    13501 gaagccctca gggaaggact gcttgaagaa gtcggggatg ccctgggtgt ggttgatgaa
    13561 ggtcttgctg ccgtagagga agctagtagc caggatgtcg aaggcgaagg ggagagggcc
    13621 gccctcgacc accttgattc tcatggtctg ggtgccctcg tagggcttgc cttcgccctc
    13681 ggatgtgcac ttgaagtgat ggttgtccac ggtgccctcc atgtacagct tcatgtgcat
    13741 gttctcctta atcagctctt cgcccttaga caccatggtg gcggtggctc cccctgaccc
    13801 tccatccaac ttaacgcgct ttgcagcagg acctccagaa ccgccgtcga gtttgacccg
    13861 tttagcggca gggaccggtg ggccaggatt ctcctcgacg tcaccgcatg ttagcagact
    13921 tcctctgccc tctcccgatc cggcaccggg cttgcgggtc atgcaccagg tgcgcggtcc
    13981 ttcgggcacc tcgacgtcgg cggtgacggt gaagccgagc cgctcgtaga aggggaggtt
    14041 gcggggcgcg gaggtctcca ggaaggcggg caccccggcg cgctcggccg cctccactcc
    14101 ggggagcacg acggcgctgc ccagaccctt gccctggtgg tcgggcgaga ctccgacggt
    14161 ggccaggaac cacgcgggct ccttgggccg gtgcggcgcc aggaggcctt ccatctgttg
    14221 ctgcgcggcc agccgggaac cgctcaactc ggccatgcgc gggccgatct cggcgaacac
    14281 cgcccccgct tcgacgctct ccggcgtggt ccagaccgcc acggcggcgc cgtcgtccgc
    14341 gacccacacc ttgccgatgt cgagcccgac gcgcgtgagg aagagttctt gcagctcggt
    14401 gacccgctcg atgtggcggt ccggatcgac ggtgtggcgc gtggcggggt agtcggcgaa
    14461 cgcggcggcg agggtgcgta cggccctggg gacgtcgtcg cgggtggcga ggcgcaccgt
    14521 gggcttgtac tcggtcatgg tgggatttaa atcacgacac ctgaaatgga agaaaaaaac
    14581 tttgaaccac tgtctgaggc ttgagaatga accaagatcc aaactcaaaa agggcaaatt
    14641 ccaaggagaa ttacatcaag tgccaagctg gcctaacttc agtctccacc cactcagtgt
    14701 ggggaaactc catcgcataa aacccctccc cccaacctaa agacgacgta ctccaaaagc
    14761 gcgagaacta atcgaggtgc ctggacggcg cccggtactc cgtggagtca catgaagcga
    14821 cggctgagga cggaaaggcc cttttccttt gtgtgggtga ctcacccgcc cgctctcccg
    14881 agcgccgcgt cctccatttt gagctccctg cagcagggcc gggaagcggc catctttccg
    14941 ctcacgcaac tggtgccgac cgggccagcc ttgccgccca gggcggggcg atacacggcg
    15001 gcgcgaggcc aggcaccaga gcaggccggc cagcttgaga ctacccccgt ccgattctcg
    15061 gtggccgcgc tcgcaggccc cgcctcgccg aacatgtgcg ctgggacgca cgggccccgt
    15121 cgccgcccgc ggccccaaaa accgaaatac cagtgtgcag atcttggccc gcatttacaa
    15181 gactatcttg ccagaaaaaa agcgtcgcag caggtcatca aaaattttaa atggctagag
    15241 acttatcgaa agcagcgaga caggcgcgaa ggtgccacca gattcgcacg cggcggcccc
    15301 agcgcccagg ccaggcctca actcaagcac gaggcgaagg ggctccttaa gcgcaaggcc
    15361 tcgaactctc ccacccactt ccaacccgaa gctcgggatc aagaatcacg tactgcagcc
    15421 aggtggaagt aattcaaggc acgcaagggc cataacccgt aaagaggcca ggcccgcggg
    15481 aaccacacac ggcacttacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca
    15541 cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt
    15601 acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg gttcaattgc
    15661 cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc cataacttcg
    15721 tataatgtat gctatacgaa gttataatat tctcactgac tccgtcctgg agttggatga
    15781 gagataatgg ccttacgttg tgccagggga gggtcgggct ggatttagca agatttacct
    15841 tctccaaaga gcggtgctgc agtggcacag ctgcccacgg aggtgggggg gtcaccgtcc
    15901 ctggaggtga tgaagaactg tggggatgtg gcactgaggg acatggccag tgggcacggt
    15961 gggtgggttg gggttggtct tggggatctt ggagggcttt tccagccttc atgatttgac
    16021 gattgtatga acatctacat ggcaattctc cagctgcctg tcccagtcct actgacccag
    16081 ctgtatctct ccaggcaagc tcttccaccc cttctgcttg catccagaca ccatcaaaca
    16141 tgcaggctca gacacaggga ccagcagtgt ctgtggcctt tttgtgctcc tctccatgct
    16201 gggttttaac ttgctctttg tccttctatc ctatcttctt atccttaagg ctgttctgaa
    16261 cgctgtgact tggagagtgt cccagagccc tcaacacctg catgtcccac gtccatgctg
    16321 tcctgcactt ccttatcccc aagatctgcc tctccgtgat gcactgaatt ggcaaacatg
    16381 tgtcacccca gaccaacaat gtcacagcaa actccccctt gataggacaa gggggaatgg
    16441 ctttacactg agacagggga ggtttgggtt ggatatgagg aggcagtttt tcccccagag
    16501 ggtggtgacg cactgaacag gttgcccaag gaggctgtgg atgccccatc cctgcaggca
    16561 ttcaaggcca ggctggatgt ggctctgggc agcctgggct gctggttgat gaccctgcac
    16621 atagcagggg gttggatctg gatgagcact gtgctccttt gcaacccagg ccgttctatg
    16681 attctgtcat tctaaatctc tctttcagcc taaagctttt tccccgtatc cccccaggtg
    16741 tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc
    16801 ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg
    16861 gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc
    16921 ctgggggctt tggggggggg ctgtccccgt gagctccacg tgcctagggc acacgaccac
    16981 aattccactg aaagcatttt aatacggaac ttgtcactcc cagggagcct ccgctcagcc
    17041 ggcagttggt tcatttcaat ccccacgaca acccttcaaa gtgcagggca gacagcaggt
    17101 ggctctgccc aggcgcctgg atcacagccc ggcctgcagc cctcacctgg gcgcggggag
    17161 accctgagga cgctcctcca ggcggcgctg gccggggcct gcggacacgg acgggcgggc
    17221 tgagctccgg gacccctccc cgcgccccgc accccgcacc ccgcaccccg caccccgcac
    17281 ccggcgctca cccgtcccag ccccgccgcc cgcagcccca gctgcaacgc agccaccgcc
    17341 gccatcgcac ccggccccgc gggcgcttcc gggacgcagg aggcatctgc atccggggcg
    17401 ccgctgagtc ccgcccagag ccccgccccc ggctccaggt tctgcgagcg gcttccgccg
    17461 ggctgctccg cgggcgcgtc ggccatgagc gagttgccgg gcgacgtgcg ggcgtttctg
    17521 cgggagcacc cgagcctgcg gctccagacg gacgcccgca aggttcgcag cgcgggaggg
    17581 gaacggagtg gcggagaagg gcgcagttgg gatgaggggc tgaggggagg gcaggggaga
    17641 ggagagggca ggggagaggg gagaggggag agcaggagag aggggaaggc aggggagagg
    17701 gcgcggcggg atcaggggag gagagggaag ggggcgcggc aggagggggc accagggagc
    17761 ggagccctgg ccctcctgac gtcctgcccg cccacgcgtc cgcaggtgag gtgcatcctg
    17821 acaggtcacg agctgccctg ccgcctgccg gagctccagg tctacacccg cggcaaaaag
    17881 taccagcggc tggtccgcgc ctccccggcc ttcgactatg cagagttcga gccgcacatc
    17941 gtgcccagca ccaagaaccc gtaggtggtc
//
