LOCUS       sequence-403331-       17976 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 17976)
  TITLE     CRISPRi by Zim3-dCas9 for iPSC Neurodegenerative Disease Initiative 
            (iNDI)
REFERENCE   2  (bases 1 to 17976)
  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: iE65 PB-Zim3-XTEN-dCas9-mApple-puro-BFP
FEATURES             Location/Qualifiers
     source          1..17976
                     /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
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             6310..6330
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6337..6357
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6373..6426
                     /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             6427..6453
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6469..6495
                     /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    7255..7843
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7308..7328)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7726..7737)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7875..8351
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(8074..8093)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   8796..8858
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8988..9005)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8988..9004)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8997..9019)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9146..9601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9233..9252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9443..9464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9627..9731
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9732..9800,9801..10592)
                     /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(9950..9969)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10763..11351
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11252..11271
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11505..11522
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11639..11660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11675..11680,11681..11698,11699..11705)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11713..11729
                     /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     11718..11740
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11737..11753
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11737..11753
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   12244..12278
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    12794..12827
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    12828..13052
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(13072..13782)
                     /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      13779..13788
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(13810..13836)
                     /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(13852..13878)
                     /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(13885..13938)
                     /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(13948..14544)
                     /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(14028..14048)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     14525..14544
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     intron          14567..15505
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(14590..14610)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(15506..15717)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    15718..15751
                     /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 cgcgcgccag 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 tcctcgaggt tatgacgtgc ccgattatgc cagcctgggc
     6301 agcggctccc ccaagaaaaa acgcaaggtg gaagatccta agaaaaagcg gaaagtggtt
     6361 aacggaagtg gcgagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct
     6421 ggcccacctg ccgctaaacg ggtcaaactc gacggcggtt ctggaggtcc tgctgcaaag
     6481 cgcgttaagt tggatgctag catggtgtcc aagggcgaag agaacaacat ggccatcatc
     6541 aaagagttca tgcggttcaa ggtgcacatg gaaggcagcg tgaacggcca cgagttcgag
     6601 attgaaggcg aaggcgaggg cagaccctac gaggcttttc agaccgccaa gctgaaagtg
     6661 accaaaggcg gccctctgcc ttttgcctgg gacattctga gccctcagtt tatgtacggc
     6721 agcaaggtgt acatcaagca ccccgccgac attcccgact acttcaagct gagcttcccc
     6781 gagggcttca gatgggagag agtgatgaac ttcgaggacg gcggcatcat ccacgtgaac
     6841 caggatagct ctctgcagga cggcgtgttc atctacaaag tgaagctgcg gggcaccaac
     6901 tttccctctg atggccccgt gatgcagaaa aagaccatgg gctgggaagc cagcgaggaa
     6961 agaatgtacc ctgaggatgg cgccctgaag tccgagatca agaagcggct gaagctgaag
     7021 gatggcggcc actatgccgc cgaagtgaaa accacctaca aggccaagaa acccgtgcag
     7081 ctgcctggcg cctacatcgt ggatattaag ctggacatcg tgtcccacaa cgaggactac
     7141 accatcgtgg aacagtacga gagagccgaa ggcagacaca gcacaggcgg aatggacgag
     7201 ctgtacaagt aggatatcgg atccatgcct aattttgagt gtattacttt cgaaaatcaa
     7261 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     7321 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     7381 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     7441 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     7501 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     7561 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     7621 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat
     7681 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     7741 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     7801 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg
     7861 gtcgacagat ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg
     7921 aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt
     7981 ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc
     8041 aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag
     8101 tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc
     8161 agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt
     8221 tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg
     8281 tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct
     8341 tccctgtcct tgtttaaacg cgatcgctta attaaggccg tggaggcctt ttccccgtat
     8401 ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg
     8461 tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga
     8521 gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg
     8581 taattacatc cctgggggct ttgggggggg gctgtccctc ataaaagttt tgttacttta
     8641 tagaagaaat tttgagtttt tgtttttttt aataaataaa taaacataaa taaattgttt
     8701 gttgaattta ttattagtat gtaagtgtaa atataataaa acttaatatc tattcaaatt
     8761 aataaataaa cctcgatata cagaccgata aaacacatgc gtcaatttta cacatgatta
     8821 tctttaacgt acgtcacaat atgattatct ttctagggtt aatctagctg cgtgttctgc
     8881 agcgtgtcga gcatcttcat ctgctccatc acgctgtaaa acacatttgc accgcgagtc
     8941 tgcccgtcct ccacgggttc aaaaacgtga atgaacgagg cgcgctcact ggccgtcgtt
     9001 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     9061 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     9121 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     9181 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     9241 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     9301 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     9361 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     9421 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     9481 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     9541 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     9601 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     9661 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     9721 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     9781 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     9841 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     9901 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     9961 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
    10021 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
    10081 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
    10141 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
    10201 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
    10261 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
    10321 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
    10381 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
    10441 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
    10501 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
    10561 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
    10621 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
    10681 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
    10741 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
    10801 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
    10861 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
    10921 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
    10981 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
    11041 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
    11101 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
    11161 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
    11221 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
    11281 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
    11341 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
    11401 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
    11461 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
    11521 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
    11581 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
    11641 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
    11701 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
    11761 acgccaagcg cgcccgccgg gtaactcacg gggtatccat gtccatttct gcggcatcca
    11821 gccaggatac ccgtcctcgc tgacgtaata tcccagcgcc gcaccgctgt cattaatctg
    11881 cacaccggca cggcagttcc ggctgtcgcc ggtattgttc gggttgctga tgcgcttcgg
    11941 gctgaccatc cggaactgtg tccggaaaag ccgcgacgaa ctggtatccc aggtggcctg
    12001 aacgaacagt tcaccgttaa aggcgtgcat ggccacacct tcccgaatca tcatggtaaa
    12061 cgtgcgtttt cgctcaacgt caatgcagca gcagtcatcc tcggcaaact ctttccatgc
    12121 cgcttcaacc tcgcgggaaa aggcacgggc ttcttcctcc ccgatgccca gatagcgcca
    12181 gcttgggcga tgactgagcc ggaaaaaaga cccgacgata tgatcctgat gcagctagat
    12241 taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat attgctctct
    12301 ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg ccgcttggag
    12361 ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact ataacgaccg
    12421 cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta actcatacga
    12481 taattatatt gttatttcat gttctactta cgtgataact tattatatat atattttctt
    12541 gttatagaat cagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc
    12601 ccgctagggg gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca
    12661 tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct
    12721 ttcctctgaa cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa
    12781 aaagccatac caaataactt cgtataatgt atgctatacg aagttatcca tagagcccac
    12841 cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac
    12901 cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta
    12961 ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca
    13021 aacaacagat ggctggcaac tagaaggcac agaattctta gcggccgcct aattaagctt
    13081 gtgccccagt ttgctaggga ggtcgcagta tctggccact gccacctcgt gctgctcgac
    13141 gtaggtctcg ttgttggcct ccttgattct ttccagtctg tagtccacat agtagacgcc
    13201 aggcatcttg aggttcttag cgggtttctt ggatctatat gtggtcttgg cgtttgcgat
    13261 cagatggctc ccgcccacga gcttcagggc catgtcgttt ctgccttcca ggccgccgtc
    13321 agcggggtac agcgtctcgg tgaaggcctc ccagccgagt gttttcttct gcatcacagg
    13381 gccgttggat gtgaagttca cccctctgat cttgacgttg tagatgaggc agccgtcctg
    13441 gaggctggtg tcctgggtag cggtcagcac gcccccgtct tcgtatgtgg tgactctctc
    13501 ccatgtgaag ccctcaggga aggactgctt gaagaagtcg gggatgccct gggtgtggtt
    13561 gatgaaggtc ttgctgccgt agaggaagct agtagccagg atgtcgaagg cgaaggggag
    13621 agggccgccc tcgaccacct tgattctcat ggtctgggtg ccctcgtagg gcttgccttc
    13681 gccctcggat gtgcacttga agtgatggtt gtccacggtg ccctccatgt acagcttcat
    13741 gtgcatgttc tccttaatca gctcttcgcc cttagacacc atggtggcgg tggctccccc
    13801 tgaccctcca tccaacttaa cgcgctttgc agcaggacct ccagaaccgc cgtcgagttt
    13861 gacccgttta gcggcaggga ccggtgggcc aggattctcc tcgacgtcac cgcatgttag
    13921 cagacttcct ctgccctctc ccgatccggc accgggcttg cgggtcatgc accaggtgcg
    13981 cggtccttcg ggcacctcga cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg
    14041 gaggttgcgg ggcgcggagg tctccaggaa ggcgggcacc ccggcgcgct cggccgcctc
    14101 cactccgggg agcacgacgg cgctgcccag acccttgccc tggtggtcgg gcgagactcc
    14161 gacggtggcc aggaaccacg cgggctcctt gggccggtgc ggcgccagga ggccttccat
    14221 ctgttgctgc gcggccagcc gggaaccgct caactcggcc atgcgcgggc cgatctcggc
    14281 gaacaccgcc cccgcttcga cgctctccgg cgtggtccag accgccacgg cggcgccgtc
    14341 gtccgcgacc cacaccttgc cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag
    14401 ctcggtgacc cgctcgatgt ggcggtccgg atcgacggtg tggcgcgtgg cggggtagtc
    14461 ggcgaacgcg gcggcgaggg tgcgtacggc cctggggacg tcgtcgcggg tggcgaggcg
    14521 caccgtgggc ttgtactcgg tcatggtggg atttaaatca cgacacctga aatggaagaa
    14581 aaaaactttg aaccactgtc tgaggcttga gaatgaacca agatccaaac tcaaaaaggg
    14641 caaattccaa ggagaattac atcaagtgcc aagctggcct aacttcagtc tccacccact
    14701 cagtgtgggg aaactccatc gcataaaacc cctcccccca acctaaagac gacgtactcc
    14761 aaaagcgcga gaactaatcg aggtgcctgg acggcgcccg gtactccgtg gagtcacatg
    14821 aagcgacggc tgaggacgga aaggcccttt tcctttgtgt gggtgactca cccgcccgct
    14881 ctcccgagcg ccgcgtcctc cattttgagc tccctgcagc agggccggga agcggccatc
    14941 tttccgctca cgcaactggt gccgaccggg ccagccttgc cgcccagggc ggggcgatac
    15001 acggcggcgc gaggccaggc accagagcag gccggccagc ttgagactac ccccgtccga
    15061 ttctcggtgg ccgcgctcgc aggccccgcc tcgccgaaca tgtgcgctgg gacgcacggg
    15121 ccccgtcgcc gcccgcggcc ccaaaaaccg aaataccagt gtgcagatct tggcccgcat
    15181 ttacaagact atcttgccag aaaaaaagcg tcgcagcagg tcatcaaaaa ttttaaatgg
    15241 ctagagactt atcgaaagca gcgagacagg cgcgaaggtg ccaccagatt cgcacgcggc
    15301 ggccccagcg cccaggccag gcctcaactc aagcacgagg cgaaggggct ccttaagcgc
    15361 aaggcctcga actctcccac ccacttccaa cccgaagctc gggatcaaga atcacgtact
    15421 gcagccaggt ggaagtaatt caaggcacgc aagggccata acccgtaaag aggccaggcc
    15481 cgcgggaacc acacacggca cttacctgtg ttctggcggc aaacccgttg cgaaaaagaa
    15541 cgttcacggc gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga
    15601 gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggttc
    15661 aattgccgac ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc tctgcccata
    15721 acttcgtata atgtatgcta tacgaagtta taatattctc actgactccg tcctggagtt
    15781 ggatgagaga taatggcctt acgttgtgcc aggggagggt cgggctggat ttagcaagat
    15841 ttaccttctc caaagagcgg tgctgcagtg gcacagctgc ccacggaggt gggggggtca
    15901 ccgtccctgg aggtgatgaa gaactgtggg gatgtggcac tgagggacat ggccagtggg
    15961 cacggtgggt gggttggggt tggtcttggg gatcttggag ggcttttcca gccttcatga
    16021 tttgacgatt gtatgaacat ctacatggca attctccagc tgcctgtccc agtcctactg
    16081 acccagctgt atctctccag gcaagctctt ccaccccttc tgcttgcatc cagacaccat
    16141 caaacatgca ggctcagaca cagggaccag cagtgtctgt ggcctttttg tgctcctctc
    16201 catgctgggt tttaacttgc tctttgtcct tctatcctat cttcttatcc ttaaggctgt
    16261 tctgaacgct gtgacttgga gagtgtccca gagccctcaa cacctgcatg tcccacgtcc
    16321 atgctgtcct gcacttcctt atccccaaga tctgcctctc cgtgatgcac tgaattggca
    16381 aacatgtgtc accccagacc aacaatgtca cagcaaactc ccccttgata ggacaagggg
    16441 gaatggcttt acactgagac aggggaggtt tgggttggat atgaggaggc agtttttccc
    16501 ccagagggtg gtgacgcact gaacaggttg cccaaggagg ctgtggatgc cccatccctg
    16561 caggcattca aggccaggct ggatgtggct ctgggcagcc tgggctgctg gttgatgacc
    16621 ctgcacatag cagggggttg gatctggatg agcactgtgc tcctttgcaa cccaggccgt
    16681 tctatgattc tgtcattcta aatctctctt tcagcctaaa gctttttccc cgtatccccc
    16741 caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga tcccgtgcca
    16801 ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc
    16861 ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag ggacgtaatt
    16921 acatccctgg gggctttggg ggggggctgt ccccgtgagc tccacgtgcc tagggcacac
    16981 gaccacaatt ccactgaaag cattttaata cggaacttgt cactcccagg gagcctccgc
    17041 tcagccggca gttggttcat ttcaatcccc acgacaaccc ttcaaagtgc agggcagaca
    17101 gcaggtggct ctgcccaggc gcctggatca cagcccggcc tgcagccctc acctgggcgc
    17161 ggggagaccc tgaggacgct cctccaggcg gcgctggccg gggcctgcgg acacggacgg
    17221 gcgggctgag ctccgggacc cctccccgcg ccccgcaccc cgcaccccgc accccgcacc
    17281 ccgcacccgg cgctcacccg tcccagcccc gccgcccgca gccccagctg caacgcagcc
    17341 accgccgcca tcgcacccgg ccccgcgggc gcttccggga cgcaggaggc atctgcatcc
    17401 ggggcgccgc tgagtcccgc ccagagcccc gcccccggct ccaggttctg cgagcggctt
    17461 ccgccgggct gctccgcggg cgcgtcggcc atgagcgagt tgccgggcga cgtgcgggcg
    17521 tttctgcggg agcacccgag cctgcggctc cagacggacg cccgcaaggt tcgcagcgcg
    17581 ggaggggaac ggagtggcgg agaagggcgc agttgggatg aggggctgag gggagggcag
    17641 gggagaggag agggcagggg agaggggaga ggggagagca ggagagaggg gaaggcaggg
    17701 gagagggcgc ggcgggatca ggggaggaga gggaaggggg cgcggcagga gggggcacca
    17761 gggagcggag ccctggccct cctgacgtcc tgcccgccca cgcgtccgca ggtgaggtgc
    17821 atcctgacag gtcacgagct gccctgccgc ctgccggagc tccaggtcta cacccgcggc
    17881 aaaaagtacc agcggctggt ccgcgcctcc ccggccttcg actatgcaga gttcgagccg
    17941 cacatcgtgc ccagcaccaa gaacccgtag gtggtc
//
