LOCUS       sequence-403330-       18058 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 18058)
  TITLE     CRISPRi by Zim3-dCas9 for iPSC Neurodegenerative Disease Initiative 
            (iNDI)
REFERENCE   2  (bases 1 to 18058)
  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: iE64
            PB-N-term_mycNLS-Zim3-XTEN-dCas9-mScarlet...
FEATURES             Location/Qualifiers
     source          1..18058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   426..488
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(618..635)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(618..634)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(627..649)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      776..1231
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(863..882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1073..1094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1257..1361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1362..1430,1431..2222)
                     /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(1580..1599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2393..2981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2882..2901
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3135..3152
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3269..3290
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3305..3310,3311..3328,3329..3335)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3343..3359
                     /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     3348..3370
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3367..3383
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3367..3383
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   3874..3908
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    4424..4457
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    4458..4682
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(4702..5412)
                     /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      5409..5418
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5440..5466)
                     /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(5482..5508)
                     /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(5515..5568)
                     /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(5578..6174)
                     /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(5658..5678)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     6155..6174
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     intron          6197..7135
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(6220..6240)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(7136..7347)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    7348..7381
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        9615..9994
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        9996..10272
                     /label=chicken beta-actin promoter
     intron          10273..11289
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     11213..11235
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     11297..11316
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             11366..11392
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             11408..11434
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             join(11852..11878,11879..11881,11882..14368,14369..14371,
                     14372..15955)
                     /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             15965..15991
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             16010..16030
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             16037..16057
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             16073..16126
                     /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             16127..16153
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             16169..16195
                     /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    16943..17531
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(16996..17016)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(17414..17425)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    17563..18039
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(17762..17781)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
ORIGIN
        1 attaaggccg tggaggcctt ttccccgtat ccccccaggt gtctgcaggc tcaaagagca
       61 gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc
      121 gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc
      181 tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg
      241 gctgtccctc ataaaagttt tgttacttta tagaagaaat tttgagtttt tgtttttttt
      301 aataaataaa taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa
      361 atataataaa acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata
      421 aaacacatgc gtcaatttta cacatgatta tctttaacgt acgtcacaat atgattatct
      481 ttctagggtt aatctagctg cgtgttctgc agcgtgtcga gcatcttcat ctgctccatc
      541 acgctgtaaa acacatttgc accgcgagtc tgcccgtcct ccacgggttc aaaaacgtga
      601 atgaacgagg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
      661 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
      721 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
      781 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      841 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      901 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
      961 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1021 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     1081 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1141 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1201 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     1261 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1321 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1381 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1441 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1501 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1561 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1621 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1681 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1741 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1801 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1861 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     1921 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     1981 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2041 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2101 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2161 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2221 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2281 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2341 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2401 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2461 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2521 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2581 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2641 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2701 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2761 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2821 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2881 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2941 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3001 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3061 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3121 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     3181 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3241 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     3301 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     3361 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcccgccgg gtaactcacg
     3421 gggtatccat gtccatttct gcggcatcca gccaggatac ccgtcctcgc tgacgtaata
     3481 tcccagcgcc gcaccgctgt cattaatctg cacaccggca cggcagttcc ggctgtcgcc
     3541 ggtattgttc gggttgctga tgcgcttcgg gctgaccatc cggaactgtg tccggaaaag
     3601 ccgcgacgaa ctggtatccc aggtggcctg aacgaacagt tcaccgttaa aggcgtgcat
     3661 ggccacacct tcccgaatca tcatggtaaa cgtgcgtttt cgctcaacgt caatgcagca
     3721 gcagtcatcc tcggcaaact ctttccatgc cgcttcaacc tcgcgggaaa aggcacgggc
     3781 ttcttcctcc ccgatgccca gatagcgcca gcttgggcga tgactgagcc ggaaaaaaga
     3841 cccgacgata tgatcctgat gcagctagat taaccctaga aagatagtct gcgtaaaatt
     3901 gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg
     3961 catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta
     4021 agtgtcactg attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt
     4081 tacgtgactt ttaagattta actcatacga taattatatt gttatttcat gttctactta
     4141 cgtgataact tattatatat atattttctt gttatagaat cagggacagc ccccccccaa
     4201 agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg
     4261 gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc
     4321 cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga
     4381 gcctgcagac acctgggggg atacggggaa aaagccatac caaataactt cgtataatgt
     4441 atgctatacg aagttatcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
     4501 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     4561 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     4621 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     4681 agaattctta gcggccgcct aattaagctt gtgccccagt ttgctaggga ggtcgcagta
     4741 tctggccact gccacctcgt gctgctcgac gtaggtctcg ttgttggcct ccttgattct
     4801 ttccagtctg tagtccacat agtagacgcc aggcatcttg aggttcttag cgggtttctt
     4861 ggatctatat gtggtcttgg cgtttgcgat cagatggctc ccgcccacga gcttcagggc
     4921 catgtcgttt ctgccttcca ggccgccgtc agcggggtac agcgtctcgg tgaaggcctc
     4981 ccagccgagt gttttcttct gcatcacagg gccgttggat gtgaagttca cccctctgat
     5041 cttgacgttg tagatgaggc agccgtcctg gaggctggtg tcctgggtag cggtcagcac
     5101 gcccccgtct tcgtatgtgg tgactctctc ccatgtgaag ccctcaggga aggactgctt
     5161 gaagaagtcg gggatgccct gggtgtggtt gatgaaggtc ttgctgccgt agaggaagct
     5221 agtagccagg atgtcgaagg cgaaggggag agggccgccc tcgaccacct tgattctcat
     5281 ggtctgggtg ccctcgtagg gcttgccttc gccctcggat gtgcacttga agtgatggtt
     5341 gtccacggtg ccctccatgt acagcttcat gtgcatgttc tccttaatca gctcttcgcc
     5401 cttagacacc atggtggcgg tggctccccc tgaccctcca tccaacttaa cgcgctttgc
     5461 agcaggacct ccagaaccgc cgtcgagttt gacccgttta gcggcaggga ccggtgggcc
     5521 aggattctcc tcgacgtcac cgcatgttag cagacttcct ctgccctctc ccgatccggc
     5581 accgggcttg cgggtcatgc accaggtgcg cggtccttcg ggcacctcga cgtcggcggt
     5641 gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg tctccaggaa
     5701 ggcgggcacc ccggcgcgct cggccgcctc cactccgggg agcacgacgg cgctgcccag
     5761 acccttgccc tggtggtcgg gcgagactcc gacggtggcc aggaaccacg cgggctcctt
     5821 gggccggtgc ggcgccagga ggccttccat ctgttgctgc gcggccagcc gggaaccgct
     5881 caactcggcc atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga cgctctccgg
     5941 cgtggtccag accgccacgg cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag
     6001 cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg
     6061 atcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc
     6121 cctggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg tcatggtggg
     6181 atttaaatca cgacacctga aatggaagaa aaaaactttg aaccactgtc tgaggcttga
     6241 gaatgaacca agatccaaac tcaaaaaggg caaattccaa ggagaattac atcaagtgcc
     6301 aagctggcct aacttcagtc tccacccact cagtgtgggg aaactccatc gcataaaacc
     6361 cctcccccca acctaaagac gacgtactcc aaaagcgcga gaactaatcg aggtgcctgg
     6421 acggcgcccg gtactccgtg gagtcacatg aagcgacggc tgaggacgga aaggcccttt
     6481 tcctttgtgt gggtgactca cccgcccgct ctcccgagcg ccgcgtcctc cattttgagc
     6541 tccctgcagc agggccggga agcggccatc tttccgctca cgcaactggt gccgaccggg
     6601 ccagccttgc cgcccagggc ggggcgatac acggcggcgc gaggccaggc accagagcag
     6661 gccggccagc ttgagactac ccccgtccga ttctcggtgg ccgcgctcgc aggccccgcc
     6721 tcgccgaaca tgtgcgctgg gacgcacggg ccccgtcgcc gcccgcggcc ccaaaaaccg
     6781 aaataccagt gtgcagatct tggcccgcat ttacaagact atcttgccag aaaaaaagcg
     6841 tcgcagcagg tcatcaaaaa ttttaaatgg ctagagactt atcgaaagca gcgagacagg
     6901 cgcgaaggtg ccaccagatt cgcacgcggc ggccccagcg cccaggccag gcctcaactc
     6961 aagcacgagg cgaaggggct ccttaagcgc aaggcctcga actctcccac ccacttccaa
     7021 cccgaagctc gggatcaaga atcacgtact gcagccaggt ggaagtaatt caaggcacgc
     7081 aagggccata acccgtaaag aggccaggcc cgcgggaacc acacacggca cttacctgtg
     7141 ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc gactactgca cttatatacg
     7201 gttctccccc accctcggga aaaaggcgga gccagtacac gacatcactt tcccagttta
     7261 ccccgcgcca ccttctctag gcaccggttc aattgccgac ccctcccccc aacttctcgg
     7321 ggactgtggg cgatgtgcgc tctgcccata acttcgtata atgtatgcta tacgaagtta
     7381 taatattctc actgactccg tcctggagtt ggatgagaga taatggcctt acgttgtgcc
     7441 aggggagggt cgggctggat ttagcaagat ttaccttctc caaagagcgg tgctgcagtg
     7501 gcacagctgc ccacggaggt gggggggtca ccgtccctgg aggtgatgaa gaactgtggg
     7561 gatgtggcac tgagggacat ggccagtggg cacggtgggt gggttggggt tggtcttggg
     7621 gatcttggag ggcttttcca gccttcatga tttgacgatt gtatgaacat ctacatggca
     7681 attctccagc tgcctgtccc agtcctactg acccagctgt atctctccag gcaagctctt
     7741 ccaccccttc tgcttgcatc cagacaccat caaacatgca ggctcagaca cagggaccag
     7801 cagtgtctgt ggcctttttg tgctcctctc catgctgggt tttaacttgc tctttgtcct
     7861 tctatcctat cttcttatcc ttaaggctgt tctgaacgct gtgacttgga gagtgtccca
     7921 gagccctcaa cacctgcatg tcccacgtcc atgctgtcct gcacttcctt atccccaaga
     7981 tctgcctctc cgtgatgcac tgaattggca aacatgtgtc accccagacc aacaatgtca
     8041 cagcaaactc ccccttgata ggacaagggg gaatggcttt acactgagac aggggaggtt
     8101 tgggttggat atgaggaggc agtttttccc ccagagggtg gtgacgcact gaacaggttg
     8161 cccaaggagg ctgtggatgc cccatccctg caggcattca aggccaggct ggatgtggct
     8221 ctgggcagcc tgggctgctg gttgatgacc ctgcacatag cagggggttg gatctggatg
     8281 agcactgtgc tcctttgcaa cccaggccgt tctatgattc tgtcattcta aatctctctt
     8341 tcagcctaaa gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag
     8401 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg
     8461 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct
     8521 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt
     8581 ccccgtgagc tccacgtgcc tagggcacac gaccacaatt ccactgaaag cattttaata
     8641 cggaacttgt cactcccagg gagcctccgc tcagccggca gttggttcat ttcaatcccc
     8701 acgacaaccc ttcaaagtgc agggcagaca gcaggtggct ctgcccaggc gcctggatca
     8761 cagcccggcc tgcagccctc acctgggcgc ggggagaccc tgaggacgct cctccaggcg
     8821 gcgctggccg gggcctgcgg acacggacgg gcgggctgag ctccgggacc cctccccgcg
     8881 ccccgcaccc cgcaccccgc accccgcacc ccgcacccgg cgctcacccg tcccagcccc
     8941 gccgcccgca gccccagctg caacgcagcc accgccgcca tcgcacccgg ccccgcgggc
     9001 gcttccggga cgcaggaggc atctgcatcc ggggcgccgc tgagtcccgc ccagagcccc
     9061 gcccccggct ccaggttctg cgagcggctt ccgccgggct gctccgcggg cgcgtcggcc
     9121 atgagcgagt tgccgggcga cgtgcgggcg tttctgcggg agcacccgag cctgcggctc
     9181 cagacggacg cccgcaaggt tcgcagcgcg ggaggggaac ggagtggcgg agaagggcgc
     9241 agttgggatg aggggctgag gggagggcag gggagaggag agggcagggg agaggggaga
     9301 ggggagagca ggagagaggg gaaggcaggg gagagggcgc ggcgggatca ggggaggaga
     9361 gggaaggggg cgcggcagga gggggcacca gggagcggag ccctggccct cctgacgtcc
     9421 tgcccgccca cgcgtccgca ggtgaggtgc atcctgacag gtcacgagct gccctgccgc
     9481 ctgccggagc tccaggtcta cacccgcggc aaaaagtacc agcggctggt ccgcgcctcc
     9541 ccggccttcg actatgcaga gttcgagccg cacatcgtgc ccagcaccaa gaacccgtag
     9601 gtggtcggcg cgccgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     9661 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     9721 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     9781 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     9841 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     9901 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     9961 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
    10021 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
    10081 attttgtgca gcgatggggg cggggggggg ggggggsgcg cgccaggcgg ggcggggcgg
    10141 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
    10201 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
    10261 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
    10321 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
    10381 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
    10441 tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg
    10501 gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc
    10561 ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg
    10621 agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc
    10681 gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa
    10741 ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc
    10801 cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg
    10861 ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc
    10921 cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg
    10981 tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg
    11041 cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa
    11101 atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct
    11161 cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc
    11221 ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt
    11281 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa
    11341 tggtacctcg cgagccgcca ccatgcctgc cgctaaacgg gtcaaactcg acggcggttc
    11401 tggaggtcct gctgcaaagc gcgttaagtt ggatggtgga tctggaggta tgaacaactc
    11461 tcagggcaga gtgaccttcg aggacgtgac cgtgaacttc acccaaggcg agtggcagag
    11521 actgaacccc gagcagagaa acctgtaccg ggacgtgatg ctggaaaact acagcaacct
    11581 ggtgtccgtc ggccagggcg agacaacaaa gcctgacgtg atcctgcggc tggaacaggg
    11641 caaagagcct tggctggaag aggaagaggt gctcggatct ggcagagccg agaagaatgg
    11701 cgacatcggc ggacagatct ggaagcccaa ggacgtgaaa gagtctctga gtggcggctc
    11761 aagtggcggg tctagtggga gtgaaacgcc agggacttca gaatcagcaa ccccggaatc
    11821 atccggcgga tcatccggtg ggtctgtata catggacaag aagtactcta tcggcctggc
    11881 catcggcacc aattctgttg gatgggccgt gatcaccgac gagtacaagg tgcccagcaa
    11941 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaatc tgatcggcgc
    12001 cctgctgttc gactctggcg aaacagccga agccaccaga ctgaagagaa ccgccagacg
    12061 gcggtacacc agaagaaaga accggatctg ctacctgcaa gagatcttca gcaacgagat
    12121 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tcgaggaaga
    12181 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gatgaggtgg cctaccacga
    12241 gaagtaccct accatctacc acctccggaa gaaactggtg gacagcaccg acaaggccga
    12301 cctgagactg atctatctgg ccctggctca catgatcaag ttccggggcc actttctgat
    12361 cgagggcgac ctgaatcctg acaacagcga cgtggacaag ctgttcatcc agctggtgca
    12421 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggagtgg atgccaaggc
    12481 catcctgtct gccagactga gcaagagcag acgcctggaa aacctgatcg ctcagctgcc
    12541 cggcgagaag aagaacggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacacc
    12601 taacttcaag agcaacttcg acctggctga ggacgccaaa ctgcagctgt ccaaggacac
    12661 ctacgacgac gacctggaca atctgctggc ccagatcggc gatcagtacg ccgacttgtt
    12721 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
    12781 cgagatcaca aaggcccctc tgagcgcctc tatgatcaag agatacgacg agcaccacca
    12841 ggatctgacc ctgctgaagg ccctcgttag acagcagctg cctgagaagt acaaagagat
    12901 tttcttcgac cagagcaaga acggctacgc cggctacatt gatggcggag ccagccaaga
    12961 ggaattttac aagttcatca agcccatcct tgagaagatg gacggcaccg aggaactgct
    13021 ggtcaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca atggctctat
    13081 ccctcaccag atccacctgg gagagctgca cgccattctg cggagacaag aggactttta
    13141 cccattcctg aaggacaacc gggaaaagat tgagaagatc ctgaccttca gaatccccta
    13201 ctacgtggga ccactggcca gaggcaatag cagattcgcc tggatgacca gaaagagcga
    13261 ggaaaccatc acaccctgga acttcgagga agtggtggac aagggcgcca gcgctcagtc
    13321 cttcatcgag cggatgacca acttcgataa gaacctgcct aacgagaagg tgctgcccaa
    13381 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
    13441 gaccgaggga atgagaaagc ccgcctttct gagcggcgag cagaaaaagg ccattgtgga
    13501 tctgctgttc aagaccaacc ggaaagtgac agtgaagcag ctgaaagagg actacttcaa
    13561 gaaaatcgag tgcttcgaca gcgtggaaat cagcggcgtg gaagatcggt tcaatgccag
    13621 cctgggcaca taccacgacc tgctgaaaat tatcaaggac aaggacttcc tggacaacga
    13681 agagaacgag gacatcctgg aagacatcgt gctgaccctg acactgtttg aggacagaga
    13741 gatgatcgag gaacggctga aaacatacgc ccacctgttc gacgacaaag tgatgaagca
    13801 actgaagcgg cggagataca ccggctgggg tagactgtcc cggaagctga tcaacggcat
    13861 ccgggataag cagtccggca agaccatcct ggactttctg aagtccgacg gcttcgccaa
    13921 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
    13981 agcccaggtg tccggccaag gcgattctct gcacgagcac attgccaacc tggccggatc
    14041 tcccgccatt aagaagggca tcctgcagac cgtgaaggtg gtggacgagc ttgtgaaagt
    14101 gatgggcaga cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
    14161 acagaagggc cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
    14221 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
    14281 gctgtacctg tactacctgc agaatggacg ggatatgtac gtggaccaag agctggacat
    14341 caaccggctg tccgactacg atgtggacgc tatcgtgccc cagtcttttc tgaaggacga
    14401 ctccatcgac aacaaggtcc tgaccagaag cgacaagaac cggggcaaga gcgataacgt
    14461 gccctccgaa gaggtggtca agaagatgaa gaactattgg cgccagctgc tgaatgccaa
    14521 gctgattacc cagcggaagt tcgacaacct gaccaaggcc gaaagaggcg gcctgagcga
    14581 actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcaccaaaca
    14641 cgtggcacag attctggact cccggatgaa cactaagtac gacgagaacg acaaactgat
    14701 ccgggaagtg aaagtcatca ccctgaagtc caagctggtg tctgatttcc ggaaggattt
    14761 ccagttctac aaagtgcgcg agatcaacaa ctaccatcac gcccacgacg cctacctgaa
    14821 tgccgttgtt ggaacagccc tgatcaagaa gtatcccaag ctggaaagcg agttcgtgta
    14881 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aagagattgg
    14941 caaggctacc gccaagtact ttttctactc caacatcatg aactttttca agacagagat
    15001 caccctggcc aacggcgaga tccggaaaag acccctgatc gagacaaacg gcgaaaccgg
    15061 ggagatcgtg tgggataagg gcagagattt tgccacagtg cggaaagtgc tgagcatgcc
    15121 ccaagtgaat atcgtgaaga aaaccgaggt gcagacaggc ggcttcagca aagagtctat
    15181 cctgcctaag cggaacagcg acaagctgat cgcccggaag aaggactggg acccaaagaa
    15241 gtacggcggc ttcgatagcc ctaccgtggc ctattctgtg ctggtggtgg ccaaagtgga
    15301 aaagggcaag tccaagaaac tcaagagcgt gaaagaactg ctcggcatca ccatcatgga
    15361 acggtccagc tttgagaaga accctattga tttccttgag gccaagggct acaaagaagt
    15421 gaaaaaggac ctgatcatca agctccccaa gtactccctg ttcgagctgg aaaatggccg
    15481 gaagcggatg ctggcttctg ctggcgaact gcagaaggga aacgaactgg ccctgcctag
    15541 caaatatgtg aactttctgt acctggccag ccactatgag aagctgaagg gcagccccga
    15601 ggacaatgag caaaagcagc tgtttgtcga gcagcacaag cactacctgg acgagatcat
    15661 cgagcagatc agcgagttct ccaagagagt gatcctggct gacgccaatc tggacaaagt
    15721 gctgtccgcc tacaacaagc accgggacaa gcctatcaga gagcaggccg agaatatcat
    15781 ccacctgttt accctgacca acctgggagc ccctgccgcc ttcaagtact tcgacaccac
    15841 catcgaccgg aagcgctaca ccagcaccaa agaggttctg gacgccacac tgatccacca
    15901 gagcatcacc ggcctgtacg agacaagaat cgacctgtct cagctcggcg gcgatcctcg
    15961 aggctatccc tatgacgtgc ccgattatgc cagcagtggc agcggctccc ccaagaaaaa
    16021 acgcaaggtg gaagatccta agaaaaagcg gaaagtggtt aacggaagtg gcgagggcag
    16081 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccacctg ccgctaaacg
    16141 ggtcaaactc gacggcggtt ctggaggtcc tgctgcaaag cgcgttaagt tggatgctag
    16201 catggaattt gtgtctaaag gggaagcagt cattaaagag ttcatgcgct tcaaagtaca
    16261 catggaagga agtatgaacg ggcacgaatt tgaaatagaa ggcgaagggg aaggtcggcc
    16321 atacgaaggg acgcaaaccg cgaagttgaa ggttacaaag ggaggaccgt tgccctttag
    16381 ctgggacatc ctgtctccac agtttatgta cggttcaaga gcttttacta agcaccccgc
    16441 tgacatcccg gactactata aacagtcatt ccccgaagga ttcaaatggg agagggtcat
    16501 gaatttcgag gacggaggtg ctgttacagt tacgcaggat acctcacttg aggatggcac
    16561 gcttatatat aaggtgaagc tccgcggtac gaactttccg ccggacggcc ccgttatgca
    16621 gaagaagacg atgggctggg aagcctctac ggaacgcctg tatcctgagg acggtgttct
    16681 gaaaggggat attaaaatgg cccttcggtt gaaagatggg ggccgctatc ttgcggactt
    16741 taagaccacc tacaaagcaa aaaagcctgt ccaaatgccc ggtgcatata acgtcgatag
    16801 aaaacttgat ataacctccc ataatgaaga ttacacagta gtcgagcagt acgaacgaag
    16861 cgaaggtcgg cactcaactg gaggtatgga tgaattgtat aagtaggata tcatgcctaa
    16921 ttttgagtgt attactttcg aaaatcaacc tctggattac aaaatttgtg aaagattgac
    16981 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
    17041 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
    17101 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
    17161 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
    17221 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
    17281 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
    17341 atcgtccttt ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt
    17401 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
    17461 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
    17521 cgcctccccg catcgatacc gagcgctggt cgacagatct acgggtggca tccctgtgac
    17581 ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc
    17641 ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg
    17701 tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg
    17761 gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc
    17821 tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc
    17881 atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc
    17941 aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg
    18001 ggattacagg cgtgaaccac tgctcccttc cctgtccttg tttaaacgcg atcgctta
//
