LOCUS       sequence-403366-       18543 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 18543)
  TITLE     CRISPRi by Zim3-dCas9 for iPSC Neurodegenerative Disease Initiative 
            (iNDI)
REFERENCE   2  (bases 1 to 18543)
  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: iF67 PB-Zim3-XTEN-dCas9-mScarlet-Bsd-Neo-mAmet...
FEATURES             Location/Qualifiers
     source          1..18543
                     /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(join(4691..5404,5405..5407,5408..5410))
                     /codon_start=1
                     /product="yellow fluorescent protein with a large Stokes 
                     shift, excitable with violet light (Ai et al., 2008)"
                     /label=mAmetrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTSGKLPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GSYRTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNVWDAYITADKQKNGIK
                     ANFKIEHNVEDGGVQLADAYQQNTPIGDGSVLLPDNHYLSFQSKLFKDPNEQRDHMVLL
                     EFVTAAGITPGMDELYK"
     primer_bind     5344..5365
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(5417..5470)
                     /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(6263..6316)
                     /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"
     intron          6770..7708
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(6793..6813)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(7709..7920)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    7921..7954
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        10188..10567
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        10569..10845
                     /label=chicken beta-actin promoter
     intron          10846..11862
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     11786..11808
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     11870..11889
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(12337..12363,12364..12366,12367..14853,14854..14856,
                     14857..16440)
                     /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             16450..16476
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             16495..16515
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             16522..16542
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             16558..16611
                     /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             16612..16638
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             16654..16680
                     /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    17428..18016
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(17481..17501)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(17899..17910)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    18048..18524
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(18247..18266)
                     /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 agaattccta ttacttatac agctcgtcca tgccgggagt gatcccggcg gcggtaacga
     4741 actccagcag gaccatgtga tcgcgctgct cgttggggtc tttgaacagc ttgctctgga
     4801 agctcaggta gtggttgtca ggcagcagca cggagccgtc gccgatgggg gtgttctgct
     4861 ggtacgcgtc ggcgagctgc acgccgccgt cctcgacgtt gtgctcgatc ttgaagttcg
     4921 ctttgatgcc gttcttctgc ttgtcggccg tgatatacgc gtcccacacg ttcatgttgt
     4981 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
     5041 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcctg tagctgccgt
     5101 cgtccttgaa ggagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
     5161 agtcgtgctg cttcatgtgg tcggggtagc gggcgaagca ctgcacgccg taagacaggg
     5221 tggtcacgag ggtgggccag ggcacgggca gcttgccgga ggtgcagatg aacttcaggg
     5281 tcagcttgcc gttggtggca tcgccctcgc cctcgccgcg cacgctgaac ttgtggccgt
     5341 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
     5401 tgctcaccat gaattcggga cctggattct cttcgacatc cccgcaagtc aggagagagc
     5461 cccgaccctc aaaaaactcg tcgaggagcc tgtagaatgc aatcctttgg ctgtcaggtg
     5521 cagcaatccc gtacaataca agaaatcggt cggcccattc cccacccagc tcctcggcaa
     5581 tatccctggt tgcaagagcg atgtcctgat agcgatcagc gacaccgagt ctcccgcagt
     5641 ctatgaaccc ggaaaatcta ccattctcga ccataatatt tggaaggcat gcatcgccat
     5701 gcgtaacaac caagtcctca ccatctggca tccgtgcttt aagtcttgca aacaactccg
     5761 cgggggcgag accctgatgt tcctcatcca agtcatcctg gtctacaaga ccagcctcca
     5821 ttcgggttct agctctctcg atccgatgtt tcgcttggtg gtcaaacggg caagtcgcag
     5881 ggtcaagcgt atggaggcgg cgcatagcat cagccataat agacaccttt tctgcagggg
     5941 caaggtggct tgagagcaag tcttgtccag gcacctcacc caacaaaagc caatctctcc
     6001 cggcctctgt cacaacgtcg agaaccgcgg cgcagggcac ccccgtggtc gcaagccaag
     6061 agaggcgagc cgcttcgtct tgaagctcat tgagggcacc gctcaaatcg gttttaacaa
     6121 aaagaactgg gcggccttga gcggagagtc gaaaaacggc ggcatcacta caccctattg
     6181 tctgctgagc ccaatcgtaa ccgaacagtc tttcaaccca tgcagccggt gatccagcat
     6241 ggagcccgtc ttgttcgatc atcggtcccg gattctcttc cacatctcca caggtaagga
     6301 gagaacctcg tccctctgta caatttcggg tatatttgag tggaatgagt tcttcaatcg
     6361 tagttttgac taacttgcca ttcatttcta ttaacacaaa acaatctggt gcatagtctg
     6421 aaatcaactc cctacacata ccacaaggac ttaccactcg aatacttcta tctacttcgt
     6481 cagaataagg gtgtctaaca gctacaatcg tgtcaaaatc cttttgtcca ttggaaactg
     6541 cactaccaat cgcaatggct tctgcacaaa cagttactcg tcctatatac gcttcaatat
     6601 gtactgccga aatgatttct cctgttttag tacgaattgc cgctcccaca tgatgtttat
     6661 tatcctcata aagcattgta atcttctctg tcgctacttc tactaattcc agatcctgct
     6721 gggagatgtt gaaggtcttc atggtggcgg cggccgcaaa tcacgacacc tgaaatggaa
     6781 gaaaaaaact ttgaaccact gtctgaggct tgagaatgaa ccaagatcca aactcaaaaa
     6841 gggcaaattc caaggagaat tacatcaagt gccaagctgg cctaacttca gtctccaccc
     6901 actcagtgtg gggaaactcc atcgcataaa acccctcccc ccaacctaaa gacgacgtac
     6961 tccaaaagcg cgagaactaa tcgaggtgcc tggacggcgc ccggtactcc gtggagtcac
     7021 atgaagcgac ggctgaggac ggaaaggccc ttttcctttg tgtgggtgac tcacccgccc
     7081 gctctcccga gcgccgcgtc ctccattttg agctccctgc agcagggccg ggaagcggcc
     7141 atctttccgc tcacgcaact ggtgccgacc gggccagcct tgccgcccag ggcggggcga
     7201 tacacggcgg cgcgaggcca ggcaccagag caggccggcc agcttgagac tacccccgtc
     7261 cgattctcgg tggccgcgct cgcaggcccc gcctcgccga acatgtgcgc tgggacgcac
     7321 gggccccgtc gccgcccgcg gccccaaaaa ccgaaatacc agtgtgcaga tcttggcccg
     7381 catttacaag actatcttgc cagaaaaaaa gcgtcgcagc aggtcatcaa aaattttaaa
     7441 tggctagaga cttatcgaaa gcagcgagac aggcgcgaag gtgccaccag attcgcacgc
     7501 ggcggcccca gcgcccaggc caggcctcaa ctcaagcacg aggcgaaggg gctccttaag
     7561 cgcaaggcct cgaactctcc cacccacttc caacccgaag ctcgggatca agaatcacgt
     7621 actgcagcca ggtggaagta attcaaggca cgcaagggcc ataacccgta aagaggccag
     7681 gcccgcggga accacacacg gcacttacct gtgttctggc ggcaaacccg ttgcgaaaaa
     7741 gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc
     7801 ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg
     7861 ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc
     7921 ataacttcgt ataatgtatg ctatacgaag ttataatatt ctcactgact ccgtcctgga
     7981 gttggatgag agataatggc cttacgttgt gccaggggag ggtcgggctg gatttagcaa
     8041 gatttacctt ctccaaagag cggtgctgca gtggcacagc tgcccacgga ggtggggggg
     8101 tcaccgtccc tggaggtgat gaagaactgt ggggatgtgg cactgaggga catggccagt
     8161 gggcacggtg ggtgggttgg ggttggtctt ggggatcttg gagggctttt ccagccttca
     8221 tgatttgacg attgtatgaa catctacatg gcaattctcc agctgcctgt cccagtccta
     8281 ctgacccagc tgtatctctc caggcaagct cttccacccc ttctgcttgc atccagacac
     8341 catcaaacat gcaggctcag acacagggac cagcagtgtc tgtggccttt ttgtgctcct
     8401 ctccatgctg ggttttaact tgctctttgt ccttctatcc tatcttctta tccttaaggc
     8461 tgttctgaac gctgtgactt ggagagtgtc ccagagccct caacacctgc atgtcccacg
     8521 tccatgctgt cctgcacttc cttatcccca agatctgcct ctccgtgatg cactgaattg
     8581 gcaaacatgt gtcaccccag accaacaatg tcacagcaaa ctcccccttg ataggacaag
     8641 ggggaatggc tttacactga gacaggggag gtttgggttg gatatgagga ggcagttttt
     8701 cccccagagg gtggtgacgc actgaacagg ttgcccaagg aggctgtgga tgccccatcc
     8761 ctgcaggcat tcaaggccag gctggatgtg gctctgggca gcctgggctg ctggttgatg
     8821 accctgcaca tagcaggggg ttggatctgg atgagcactg tgctcctttg caacccaggc
     8881 cgttctatga ttctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc
     8941 ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg
     9001 ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc
     9061 gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta
     9121 attacatccc tgggggcttt gggggggggc tgtccccgtg agctccacgt gcctagggca
     9181 cacgaccaca attccactga aagcatttta atacggaact tgtcactccc agggagcctc
     9241 cgctcagccg gcagttggtt catttcaatc cccacgacaa cccttcaaag tgcagggcag
     9301 acagcaggtg gctctgccca ggcgcctgga tcacagcccg gcctgcagcc ctcacctggg
     9361 cgcggggaga ccctgaggac gctcctccag gcggcgctgg ccggggcctg cggacacgga
     9421 cgggcgggct gagctccggg acccctcccc gcgccccgca ccccgcaccc cgcaccccgc
     9481 accccgcacc cggcgctcac ccgtcccagc cccgccgccc gcagccccag ctgcaacgca
     9541 gccaccgccg ccatcgcacc cggccccgcg ggcgcttccg ggacgcagga ggcatctgca
     9601 tccggggcgc cgctgagtcc cgcccagagc cccgcccccg gctccaggtt ctgcgagcgg
     9661 cttccgccgg gctgctccgc gggcgcgtcg gccatgagcg agttgccggg cgacgtgcgg
     9721 gcgtttctgc gggagcaccc gagcctgcgg ctccagacgg acgcccgcaa ggttcgcagc
     9781 gcgggagggg aacggagtgg cggagaaggg cgcagttggg atgaggggct gaggggaggg
     9841 caggggagag gagagggcag gggagagggg agaggggaga gcaggagaga ggggaaggca
     9901 ggggagaggg cgcggcggga tcaggggagg agagggaagg gggcgcggca ggagggggca
     9961 ccagggagcg gagccctggc cctcctgacg tcctgcccgc ccacgcgtcc gcaggtgagg
    10021 tgcatcctga caggtcacga gctgccctgc cgcctgccgg agctccaggt ctacacccgc
    10081 ggcaaaaagt accagcggct ggtccgcgcc tccccggcct tcgactatgc agagttcgag
    10141 ccgcacatcg tgcccagcac caagaacccg taggtggtcg gcgcgccgac attgattatt
    10201 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
    10261 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
    10321 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
    10381 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
    10441 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
    10501 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
    10561 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
    10621 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
    10681 gggggggggs gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
    10741 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
    10801 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
    10861 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
    10921 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
    10981 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
    11041 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
    11101 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
    11161 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
    11221 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
    11281 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
    11341 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
    11401 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
    11461 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
    11521 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
    11581 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
    11641 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
    11701 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
    11761 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
    11821 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
    11881 gttattgtgc tgtctcatca ttttggcaaa gaaggtacct cgcgagccgc caccatgaac
    11941 aactctcagg gcagagtgac cttcgaggac gtgaccgtga acttcaccca aggcgagtgg
    12001 cagagactga accccgagca gagaaacctg taccgggacg tgatgctgga aaactacagc
    12061 aacctggtgt ccgtcggcca gggcgagaca acaaagcctg acgtgatcct gcggctggaa
    12121 cagggcaaag agccttggct ggaagaggaa gaggtgctcg gatctggcag agccgagaag
    12181 aatggcgaca tcggcggaca gatctggaag cccaaggacg tgaaagagtc tctgagtggc
    12241 ggctcaagtg gcgggtctag tgggagtgaa acgccaggga cttcagaatc agcaaccccg
    12301 gaatcatccg gcggatcatc cggtgggtct gtatacatgg acaagaagta ctctatcggc
    12361 ctggccatcg gcaccaattc tgttggatgg gccgtgatca ccgacgagta caaggtgccc
    12421 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaatctgatc
    12481 ggcgccctgc tgttcgactc tggcgaaaca gccgaagcca ccagactgaa gagaaccgcc
    12541 agacggcggt acaccagaag aaagaaccgg atctgctacc tgcaagagat cttcagcaac
    12601 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtcgag
    12661 gaagataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggatga ggtggcctac
    12721 cacgagaagt accctaccat ctaccacctc cggaagaaac tggtggacag caccgacaag
    12781 gccgacctga gactgatcta tctggccctg gctcacatga tcaagttccg gggccacttt
    12841 ctgatcgagg gcgacctgaa tcctgacaac agcgacgtgg acaagctgtt catccagctg
    12901 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg agtggatgcc
    12961 aaggccatcc tgtctgccag actgagcaag agcagacgcc tggaaaacct gatcgctcag
    13021 ctgcccggcg agaagaagaa cggcctgttc ggaaacctga ttgccctgag cctgggcctg
    13081 acacctaact tcaagagcaa cttcgacctg gctgaggacg ccaaactgca gctgtccaag
    13141 gacacctacg acgacgacct ggacaatctg ctggcccaga tcggcgatca gtacgccgac
    13201 ttgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
    13261 aacaccgaga tcacaaaggc ccctctgagc gcctctatga tcaagagata cgacgagcac
    13321 caccaggatc tgaccctgct gaaggccctc gttagacagc agctgcctga gaagtacaaa
    13381 gagattttct tcgaccagag caagaacggc tacgccggct acattgatgg cggagccagc
    13441 caagaggaat tttacaagtt catcaagccc atccttgaga agatggacgg caccgaggaa
    13501 ctgctggtca agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaatggc
    13561 tctatccctc accagatcca cctgggagag ctgcacgcca ttctgcggag acaagaggac
    13621 ttttacccat tcctgaagga caaccgggaa aagattgaga agatcctgac cttcagaatc
    13681 ccctactacg tgggaccact ggccagaggc aatagcagat tcgcctggat gaccagaaag
    13741 agcgaggaaa ccatcacacc ctggaacttc gaggaagtgg tggacaaggg cgccagcgct
    13801 cagtccttca tcgagcggat gaccaacttc gataagaacc tgcctaacga gaaggtgctg
    13861 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
    13921 tacgtgaccg agggaatgag aaagcccgcc tttctgagcg gcgagcagaa aaaggccatt
    13981 gtggatctgc tgttcaagac caaccggaaa gtgacagtga agcagctgaa agaggactac
    14041 ttcaagaaaa tcgagtgctt cgacagcgtg gaaatcagcg gcgtggaaga tcggttcaat
    14101 gccagcctgg gcacatacca cgacctgctg aaaattatca aggacaagga cttcctggac
    14161 aacgaagaga acgaggacat cctggaagac atcgtgctga ccctgacact gtttgaggac
    14221 agagagatga tcgaggaacg gctgaaaaca tacgcccacc tgttcgacga caaagtgatg
    14281 aagcaactga agcggcggag atacaccggc tggggtagac tgtcccggaa gctgatcaac
    14341 ggcatccggg ataagcagtc cggcaagacc atcctggact ttctgaagtc cgacggcttc
    14401 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
    14461 cagaaagccc aggtgtccgg ccaaggcgat tctctgcacg agcacattgc caacctggcc
    14521 ggatctcccg ccattaagaa gggcatcctg cagaccgtga aggtggtgga cgagcttgtg
    14581 aaagtgatgg gcagacacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
    14641 accacacaga agggccagaa gaacagccgc gagagaatga agcggatcga agagggcatc
    14701 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
    14761 gagaagctgt acctgtacta cctgcagaat ggacgggata tgtacgtgga ccaagagctg
    14821 gacatcaacc ggctgtccga ctacgatgtg gacgctatcg tgccccagtc ttttctgaag
    14881 gacgactcca tcgacaacaa ggtcctgacc agaagcgaca agaaccgggg caagagcgat
    14941 aacgtgccct ccgaagaggt ggtcaagaag atgaagaact attggcgcca gctgctgaat
    15001 gccaagctga ttacccagcg gaagttcgac aacctgacca aggccgaaag aggcggcctg
    15061 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcacc
    15121 aaacacgtgg cacagattct ggactcccgg atgaacacta agtacgacga gaacgacaaa
    15181 ctgatccggg aagtgaaagt catcaccctg aagtccaagc tggtgtctga tttccggaag
    15241 gatttccagt tctacaaagt gcgcgagatc aacaactacc atcacgccca cgacgcctac
    15301 ctgaatgccg ttgttggaac agccctgatc aagaagtatc ccaagctgga aagcgagttc
    15361 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaagag
    15421 attggcaagg ctaccgccaa gtactttttc tactccaaca tcatgaactt tttcaagaca
    15481 gagatcaccc tggccaacgg cgagatccgg aaaagacccc tgatcgagac aaacggcgaa
    15541 accggggaga tcgtgtggga taagggcaga gattttgcca cagtgcggaa agtgctgagc
    15601 atgccccaag tgaatatcgt gaagaaaacc gaggtgcaga caggcggctt cagcaaagag
    15661 tctatcctgc ctaagcggaa cagcgacaag ctgatcgccc ggaagaagga ctgggaccca
    15721 aagaagtacg gcggcttcga tagccctacc gtggcctatt ctgtgctggt ggtggccaaa
    15781 gtggaaaagg gcaagtccaa gaaactcaag agcgtgaaag aactgctcgg catcaccatc
    15841 atggaacggt ccagctttga gaagaaccct attgatttcc ttgaggccaa gggctacaaa
    15901 gaagtgaaaa aggacctgat catcaagctc cccaagtact ccctgttcga gctggaaaat
    15961 ggccggaagc ggatgctggc ttctgctggc gaactgcaga agggaaacga actggccctg
    16021 cctagcaaat atgtgaactt tctgtacctg gccagccact atgagaagct gaagggcagc
    16081 cccgaggaca atgagcaaaa gcagctgttt gtcgagcagc acaagcacta cctggacgag
    16141 atcatcgagc agatcagcga gttctccaag agagtgatcc tggctgacgc caatctggac
    16201 aaagtgctgt ccgcctacaa caagcaccgg gacaagccta tcagagagca ggccgagaat
    16261 atcatccacc tgtttaccct gaccaacctg ggagcccctg ccgccttcaa gtacttcgac
    16321 accaccatcg accggaagcg ctacaccagc accaaagagg ttctggacgc cacactgatc
    16381 caccagagca tcaccggcct gtacgagaca agaatcgacc tgtctcagct cggcggcgat
    16441 cctcgaggct atccctatga cgtgcccgat tatgccagca gtggcagcgg ctcccccaag
    16501 aaaaaacgca aggtggaaga tcctaagaaa aagcggaaag tggttaacgg aagtggcgag
    16561 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc acctgccgct
    16621 aaacgggtca aactcgacgg cggttctgga ggtcctgctg caaagcgcgt taagttggat
    16681 gctagcatgg aatttgtgtc taaaggggaa gcagtcatta aagagttcat gcgcttcaaa
    16741 gtacacatgg aaggaagtat gaacgggcac gaatttgaaa tagaaggcga aggggaaggt
    16801 cggccatacg aagggacgca aaccgcgaag ttgaaggtta caaagggagg accgttgccc
    16861 tttagctggg acatcctgtc tccacagttt atgtacggtt caagagcttt tactaagcac
    16921 cccgctgaca tcccggacta ctataaacag tcattccccg aaggattcaa atgggagagg
    16981 gtcatgaatt tcgaggacgg aggtgctgtt acagttacgc aggatacctc acttgaggat
    17041 ggcacgctta tatataaggt gaagctccgc ggtacgaact ttccgccgga cggccccgtt
    17101 atgcagaaga agacgatggg ctgggaagcc tctacggaac gcctgtatcc tgaggacggt
    17161 gttctgaaag gggatattaa aatggccctt cggttgaaag atgggggccg ctatcttgcg
    17221 gactttaaga ccacctacaa agcaaaaaag cctgtccaaa tgcccggtgc atataacgtc
    17281 gatagaaaac ttgatataac ctcccataat gaagattaca cagtagtcga gcagtacgaa
    17341 cgaagcgaag gtcggcactc aactggaggt atggatgaat tgtataagta ggatatcatg
    17401 cctaattttg agtgtattac tttcgaaaat caacctctgg attacaaaat ttgtgaaaga
    17461 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
    17521 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
    17581 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
    17641 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
    17701 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
    17761 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
    17821 aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg
    17881 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
    17941 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
    18001 tgggccgcct ccccgcatcg ataccgagcg ctggtcgaca gatctacggg tggcatccct
    18061 gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc
    18121 ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta
    18181 tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct
    18241 gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct
    18301 ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg
    18361 catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat
    18421 tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat
    18481 tgctgggatt acaggcgtga accactgctc ccttccctgt ccttgtttaa acgcgatcgc
    18541 tta
//
