LOCUS       sequence-403322-       18966 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 18966)
  TITLE     CRISPRi by Zim3-dCas9 for iPSC Neurodegenerative Disease Initiative 
            (iNDI)
REFERENCE   2  (bases 1 to 18966)
  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: iF51 PB-Zim3-dCas9-mScarlet-Hygro-Neo-SnapTag
FEATURES             Location/Qualifiers
     source          1..18966
                     /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(5243..5296)
                     /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(6089..6142)
                     /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(6143..7165)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     intron          7193..8131
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(7216..7236)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(8132..8343)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    8344..8377
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        10611..10990
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        10992..11268
                     /label=chicken beta-actin promoter
     intron          11269..12285
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     12209..12231
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     12293..12312
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(12760..12786,12787..12789,12790..15276,15277..15279,
                     15280..16863)
                     /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             16873..16899
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             16918..16938
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             16945..16965
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             16981..17034
                     /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             17035..17061
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             17077..17103
                     /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    17851..18439
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(17904..17924)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(18322..18333)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    18471..18947
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(18670..18689)
                     /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 acccagcccg ggttttccaa ggcgatgccc ctcatgagcc aggagccatt
     4741 ccttcacagc aagtcctcct tcgtatcctc cgacgtccaa gtcgccttgg accacccgat
     4801 ggcacggaat gagtattggg actgggtttc cagacaacgc cgtttttact gccgccgttg
     4861 cggcaggatt cccagccaag gccgccagat gactatagct gattacctcg ccaaatttca
     4921 ctaccttcaa gagcttccac aatacctgtc ttgtgaatga ctcttgctgg aagacaggat
     4981 gatggagcgc cggtactggg aactcttcta tggcctcggg ttggtggaag taggcgttca
     5041 accaggctgt cgcttgcatg agaggttctg gaccgccaag taccgcggcg ggcgcaggca
     5101 cctctaccgc gtccgcggcg ctggtcccct tcccaaggaa aataatccgg tgaagcccct
     5161 gttcacatcc tgacagttcc agcttaccaa gaggactgtc caaggtagtg cgtttcatct
     5221 cgcagtcctt atccatgaat tcgggacctg gattctcttc gacatccccg caagtcagga
     5281 gagagccccg accctcaaaa aactcgtcga ggagcctgta gaatgcaatc ctttggctgt
     5341 caggtgcagc aatcccgtac aatacaagaa atcggtcggc ccattcccca cccagctcct
     5401 cggcaatatc cctggttgca agagcgatgt cctggtaccg gtcagcgaca ccgagtctcc
     5461 cgcagtctat gaacccggaa aatctaccat tctcgaccat aatatttgga aggcatgcat
     5521 cgccatgcgt aacaaccaag tcctcaccat ctggcatccg tgctttaagt cttgcaaaca
     5581 actccgcggg ggcgagaccc tgatgttcct catccaagtc atcctggtct acaagaccag
     5641 cctccattcg ggttctagct ctctcgatcc gatgtttcgc ttggtggtca aacgggcaag
     5701 tcgcagggtc aagcgtatgg aggcggcgca tagcatcagc cataatagac accttttctg
     5761 caggggcaag gtggcttgag agcaagtctt gtccaggtac ctcacccaac aaaagccaat
     5821 ctctcccggc ctctgtcaca acgtcgagaa ccgcggcgca gggtaccccc gtggtcgcaa
     5881 gccaagagag gcgagccgct tcgtcttgaa gctcattgag ggcaccgctc aaatcggttt
     5941 taacaaaaag aactgggcgg ccttgagcgg agagtcgaaa aacggcggca tcactacacc
     6001 ctattgtctg ctgagcccaa tcgtaaccga acagtctttc aacccatgca gccggtgatc
     6061 cagcatggag cccgtcttgt tcgatcatcg gtcccggatt ctcttccaca tctccacagg
     6121 taaggagaga acctcgtccc tcctctttag ctcgcgggcg agtagaaggc cggcgattac
     6181 ctgaatccgc caatacctcg acgcagccgt cggtccatac agccgcactc cttcgagcta
     6241 tctgtgttct gccgacggta ccggctccac tccttactat tgcatcacac ctaccttgag
     6301 cccacgccgc atcatcgaag ttcccatcta cgagtgactg atagagctga tccagtccaa
     6361 tcctcaacat gtacgcgcgc aatctcggac tacctgccaa ttcaggatgg cgtcgctcaa
     6421 aatacctcgt ttgttgctcc atgcatgcga gccagggccg ccagaaaaat atatttgcta
     6481 cctcatattg tgagtcgccg aacatcgcct ctgaccagtc tataacagcg gttatccgcc
     6541 cattatccgt caaaacgtta tttgaaccga agtcagcgtg aaccaagtgg cgtacctcag
     6601 gacagtcttc tgcccagagc atcagttcat caagcgcttg cgcgactgag gcagaaaccg
     6661 tatcatccat taccgtttgc caatggtaga cgtgcggatc tgcaatggcg cagatgaagt
     6721 ctctccaagt cgtgtattgg cctatgcctt gcggaccaaa ggggccgaat ccggacgtct
     6781 gagagagatc agccgcggct atggcatcca tagcttcagc gaccggttgg agaacggctg
     6841 gaagctcggt ttccggcaga tcctgaagtg taaccccctg agcgcggcga ctgatgcagt
     6901 aagtcaggct ctcagaaaat tcaccaatat ccaatacttc gggaatgggg agtgcagcgg
     6961 aagcgaagtg tctatataca tacctgtcct tgtagaatcc gtctgcgcag gagttcaccc
     7021 tgaggacata gcccctcccc cctacatcaa aggagaaggc tcgggactcc tctccttctg
     7081 aaagctgcat cagatcgcta actgaatcaa atttttctat caagaacttt tcaacagatg
     7141 ttgcagtcaa ctctggcttc ttcatggtgg cggcggccgc aaatcacgac acctgaaatg
     7201 gaagaaaaaa actttgaacc actgtctgag gcttgagaat gaaccaagat ccaaactcaa
     7261 aaagggcaaa ttccaaggag aattacatca agtgccaagc tggcctaact tcagtctcca
     7321 cccactcagt gtggggaaac tccatcgcat aaaacccctc cccccaacct aaagacgacg
     7381 tactccaaaa gcgcgagaac taatcgaggt gcctggacgg cgcccggtac tccgtggagt
     7441 cacatgaagc gacggctgag gacggaaagg cccttttcct ttgtgtgggt gactcacccg
     7501 cccgctctcc cgagcgccgc gtcctccatt ttgagctccc tgcagcaggg ccgggaagcg
     7561 gccatctttc cgctcacgca actggtgccg accgggccag ccttgccgcc cagggcgggg
     7621 cgatacacgg cggcgcgagg ccaggcacca gagcaggccg gccagcttga gactaccccc
     7681 gtccgattct cggtggccgc gctcgcaggc cccgcctcgc cgaacatgtg cgctgggacg
     7741 cacgggcccc gtcgccgccc gcggccccaa aaaccgaaat accagtgtgc agatcttggc
     7801 ccgcatttac aagactatct tgccagaaaa aaagcgtcgc agcaggtcat caaaaatttt
     7861 aaatggctag agacttatcg aaagcagcga gacaggcgcg aaggtgccac cagattcgca
     7921 cgcggcggcc ccagcgccca ggccaggcct caactcaagc acgaggcgaa ggggctcctt
     7981 aagcgcaagg cctcgaactc tcccacccac ttccaacccg aagctcggga tcaagaatca
     8041 cgtactgcag ccaggtggaa gtaattcaag gcacgcaagg gccataaccc gtaaagaggc
     8101 caggcccgcg ggaaccacac acggcactta cctgtgttct ggcggcaaac ccgttgcgaa
     8161 aaagaacgtt cacggcgact actgcactta tatacggttc tcccccaccc tcgggaaaaa
     8221 ggcggagcca gtacacgaca tcactttccc agtttacccc gcgccacctt ctctaggcac
     8281 cggttcaatt gccgacccct ccccccaact tctcggggac tgtgggcgat gtgcgctctg
     8341 cccataactt cgtataatgt atgctatacg aagttataat attctcactg actccgtcct
     8401 ggagttggat gagagataat ggccttacgt tgtgccaggg gagggtcggg ctggatttag
     8461 caagatttac cttctccaaa gagcggtgct gcagtggcac agctgcccac ggaggtgggg
     8521 gggtcaccgt ccctggaggt gatgaagaac tgtggggatg tggcactgag ggacatggcc
     8581 agtgggcacg gtgggtgggt tggggttggt cttggggatc ttggagggct tttccagcct
     8641 tcatgatttg acgattgtat gaacatctac atggcaattc tccagctgcc tgtcccagtc
     8701 ctactgaccc agctgtatct ctccaggcaa gctcttccac cccttctgct tgcatccaga
     8761 caccatcaaa catgcaggct cagacacagg gaccagcagt gtctgtggcc tttttgtgct
     8821 cctctccatg ctgggtttta acttgctctt tgtccttcta tcctatcttc ttatccttaa
     8881 ggctgttctg aacgctgtga cttggagagt gtcccagagc cctcaacacc tgcatgtccc
     8941 acgtccatgc tgtcctgcac ttccttatcc ccaagatctg cctctccgtg atgcactgaa
     9001 ttggcaaaca tgtgtcaccc cagaccaaca atgtcacagc aaactccccc ttgataggac
     9061 aagggggaat ggctttacac tgagacaggg gaggtttggg ttggatatga ggaggcagtt
     9121 tttcccccag agggtggtga cgcactgaac aggttgccca aggaggctgt ggatgcccca
     9181 tccctgcagg cattcaaggc caggctggat gtggctctgg gcagcctggg ctgctggttg
     9241 atgaccctgc acatagcagg gggttggatc tggatgagca ctgtgctcct ttgcaaccca
     9301 ggccgttcta tgattctgtc attctaaatc tctctttcag cctaaagctt tttccccgta
     9361 tccccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc
     9421 gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg
     9481 agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac
     9541 gtaattacat ccctgggggc tttggggggg ggctgtcccc gtgagctcca cgtgcctagg
     9601 gcacacgacc acaattccac tgaaagcatt ttaatacgga acttgtcact cccagggagc
     9661 ctccgctcag ccggcagttg gttcatttca atccccacga caacccttca aagtgcaggg
     9721 cagacagcag gtggctctgc ccaggcgcct ggatcacagc ccggcctgca gccctcacct
     9781 gggcgcgggg agaccctgag gacgctcctc caggcggcgc tggccggggc ctgcggacac
     9841 ggacgggcgg gctgagctcc gggacccctc cccgcgcccc gcaccccgca ccccgcaccc
     9901 cgcaccccgc acccggcgct cacccgtccc agccccgccg cccgcagccc cagctgcaac
     9961 gcagccaccg ccgccatcgc acccggcccc gcgggcgctt ccgggacgca ggaggcatct
    10021 gcatccgggg cgccgctgag tcccgcccag agccccgccc ccggctccag gttctgcgag
    10081 cggcttccgc cgggctgctc cgcgggcgcg tcggccatga gcgagttgcc gggcgacgtg
    10141 cgggcgtttc tgcgggagca cccgagcctg cggctccaga cggacgcccg caaggttcgc
    10201 agcgcgggag gggaacggag tggcggagaa gggcgcagtt gggatgaggg gctgagggga
    10261 gggcagggga gaggagaggg caggggagag gggagagggg agagcaggag agaggggaag
    10321 gcaggggaga gggcgcggcg ggatcagggg aggagaggga agggggcgcg gcaggagggg
    10381 gcaccaggga gcggagccct ggccctcctg acgtcctgcc cgcccacgcg tccgcaggtg
    10441 aggtgcatcc tgacaggtca cgagctgccc tgccgcctgc cggagctcca ggtctacacc
    10501 cgcggcaaaa agtaccagcg gctggtccgc gcctccccgg ccttcgacta tgcagagttc
    10561 gagccgcaca tcgtgcccag caccaagaac ccgtaggtgg tcggcgcgcc gacattgatt
    10621 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
    10681 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
    10741 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
    10801 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
    10861 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
    10921 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
    10981 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
    11041 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
    11101 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
    11161 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
    11221 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
    11281 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
    11341 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
    11401 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
    11461 ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc
    11521 gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc
    11581 gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg
    11641 cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg
    11701 tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga
    11761 gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc
    11821 gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg
    11881 gccggggagg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc
    11941 gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct
    12001 ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
    12061 cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
    12121 tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc
    12181 tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
    12241 agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg
    12301 ctggttattg tgctgtctca tcattttggc aaagaaggta cctcgcgagc cgccaccatg
    12361 aacaactctc agggcagagt gaccttcgag gacgtgaccg tgaacttcac ccaaggcgag
    12421 tggcagagac tgaaccccga gcagagaaac ctgtaccggg acgtgatgct ggaaaactac
    12481 agcaacctgg tgtccgtcgg ccagggcgag acaacaaagc ctgacgtgat cctgcggctg
    12541 gaacagggca aagagccttg gctggaagag gaagaggtgc tcggatctgg cagagccgag
    12601 aagaatggcg acatcggcgg acagatctgg aagcccaagg acgtgaaaga gtctctgagt
    12661 ggcggctcaa gtggcgggtc tagtgggagt gaaacgccag ggacttcaga atcagcaacc
    12721 ccggaatcat ccggcggatc atccggtggg tctgtataca tggacaagaa gtactctatc
    12781 ggcctggcca tcggcaccaa ttctgttgga tgggccgtga tcaccgacga gtacaaggtg
    12841 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaatctg
    12901 atcggcgccc tgctgttcga ctctggcgaa acagccgaag ccaccagact gaagagaacc
    12961 gccagacggc ggtacaccag aagaaagaac cggatctgct acctgcaaga gatcttcagc
    13021 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtc
    13081 gaggaagata agaagcacga gcggcacccc atcttcggca acatcgtgga tgaggtggcc
    13141 taccacgaga agtaccctac catctaccac ctccggaaga aactggtgga cagcaccgac
    13201 aaggccgacc tgagactgat ctatctggcc ctggctcaca tgatcaagtt ccggggccac
    13261 tttctgatcg agggcgacct gaatcctgac aacagcgacg tggacaagct gttcatccag
    13321 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggagtggat
    13381 gccaaggcca tcctgtctgc cagactgagc aagagcagac gcctggaaaa cctgatcgct
    13441 cagctgcccg gcgagaagaa gaacggcctg ttcggaaacc tgattgccct gagcctgggc
    13501 ctgacaccta acttcaagag caacttcgac ctggctgagg acgccaaact gcagctgtcc
    13561 aaggacacct acgacgacga cctggacaat ctgctggccc agatcggcga tcagtacgcc
    13621 gacttgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
    13681 gtgaacaccg agatcacaaa ggcccctctg agcgcctcta tgatcaagag atacgacgag
    13741 caccaccagg atctgaccct gctgaaggcc ctcgttagac agcagctgcc tgagaagtac
    13801 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga tggcggagcc
    13861 agccaagagg aattttacaa gttcatcaag cccatccttg agaagatgga cggcaccgag
    13921 gaactgctgg tcaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaat
    13981 ggctctatcc ctcaccagat ccacctggga gagctgcacg ccattctgcg gagacaagag
    14041 gacttttacc cattcctgaa ggacaaccgg gaaaagattg agaagatcct gaccttcaga
    14101 atcccctact acgtgggacc actggccaga ggcaatagca gattcgcctg gatgaccaga
    14161 aagagcgagg aaaccatcac accctggaac ttcgaggaag tggtggacaa gggcgccagc
    14221 gctcagtcct tcatcgagcg gatgaccaac ttcgataaga acctgcctaa cgagaaggtg
    14281 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
    14341 aaatacgtga ccgagggaat gagaaagccc gcctttctga gcggcgagca gaaaaaggcc
    14401 attgtggatc tgctgttcaa gaccaaccgg aaagtgacag tgaagcagct gaaagaggac
    14461 tacttcaaga aaatcgagtg cttcgacagc gtggaaatca gcggcgtgga agatcggttc
    14521 aatgccagcc tgggcacata ccacgacctg ctgaaaatta tcaaggacaa ggacttcctg
    14581 gacaacgaag agaacgagga catcctggaa gacatcgtgc tgaccctgac actgtttgag
    14641 gacagagaga tgatcgagga acggctgaaa acatacgccc acctgttcga cgacaaagtg
    14701 atgaagcaac tgaagcggcg gagatacacc ggctggggta gactgtcccg gaagctgatc
    14761 aacggcatcc gggataagca gtccggcaag accatcctgg actttctgaa gtccgacggc
    14821 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
    14881 atccagaaag cccaggtgtc cggccaaggc gattctctgc acgagcacat tgccaacctg
    14941 gccggatctc ccgccattaa gaagggcatc ctgcagaccg tgaaggtggt ggacgagctt
    15001 gtgaaagtga tgggcagaca caagcccgag aacatcgtga tcgaaatggc cagagagaac
    15061 cagaccacac agaagggcca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
    15121 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
    15181 aacgagaagc tgtacctgta ctacctgcag aatggacggg atatgtacgt ggaccaagag
    15241 ctggacatca accggctgtc cgactacgat gtggacgcta tcgtgcccca gtcttttctg
    15301 aaggacgact ccatcgacaa caaggtcctg accagaagcg acaagaaccg gggcaagagc
    15361 gataacgtgc cctccgaaga ggtggtcaag aagatgaaga actattggcg ccagctgctg
    15421 aatgccaagc tgattaccca gcggaagttc gacaacctga ccaaggccga aagaggcggc
    15481 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
    15541 accaaacacg tggcacagat tctggactcc cggatgaaca ctaagtacga cgagaacgac
    15601 aaactgatcc gggaagtgaa agtcatcacc ctgaagtcca agctggtgtc tgatttccgg
    15661 aaggatttcc agttctacaa agtgcgcgag atcaacaact accatcacgc ccacgacgcc
    15721 tacctgaatg ccgttgttgg aacagccctg atcaagaagt atcccaagct ggaaagcgag
    15781 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcaa
    15841 gagattggca aggctaccgc caagtacttt ttctactcca acatcatgaa ctttttcaag
    15901 acagagatca ccctggccaa cggcgagatc cggaaaagac ccctgatcga gacaaacggc
    15961 gaaaccgggg agatcgtgtg ggataagggc agagattttg ccacagtgcg gaaagtgctg
    16021 agcatgcccc aagtgaatat cgtgaagaaa accgaggtgc agacaggcgg cttcagcaaa
    16081 gagtctatcc tgcctaagcg gaacagcgac aagctgatcg cccggaagaa ggactgggac
    16141 ccaaagaagt acggcggctt cgatagccct accgtggcct attctgtgct ggtggtggcc
    16201 aaagtggaaa agggcaagtc caagaaactc aagagcgtga aagaactgct cggcatcacc
    16261 atcatggaac ggtccagctt tgagaagaac cctattgatt tccttgaggc caagggctac
    16321 aaagaagtga aaaaggacct gatcatcaag ctccccaagt actccctgtt cgagctggaa
    16381 aatggccgga agcggatgct ggcttctgct ggcgaactgc agaagggaaa cgaactggcc
    16441 ctgcctagca aatatgtgaa ctttctgtac ctggccagcc actatgagaa gctgaagggc
    16501 agccccgagg acaatgagca aaagcagctg tttgtcgagc agcacaagca ctacctggac
    16561 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggctga cgccaatctg
    16621 gacaaagtgc tgtccgccta caacaagcac cgggacaagc ctatcagaga gcaggccgag
    16681 aatatcatcc acctgtttac cctgaccaac ctgggagccc ctgccgcctt caagtacttc
    16741 gacaccacca tcgaccggaa gcgctacacc agcaccaaag aggttctgga cgccacactg
    16801 atccaccaga gcatcaccgg cctgtacgag acaagaatcg acctgtctca gctcggcggc
    16861 gatcctcgag gctatcccta tgacgtgccc gattatgcca gcagtggcag cggctccccc
    16921 aagaaaaaac gcaaggtgga agatcctaag aaaaagcgga aagtggttaa cggaagtggc
    16981 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccacctgcc
    17041 gctaaacggg tcaaactcga cggcggttct ggaggtcctg ctgcaaagcg cgttaagttg
    17101 gatgctagca tggaatttgt gtctaaaggg gaagcagtca ttaaagagtt catgcgcttc
    17161 aaagtacaca tggaaggaag tatgaacggg cacgaatttg aaatagaagg cgaaggggaa
    17221 ggtcggccat acgaagggac gcaaaccgcg aagttgaagg ttacaaaggg aggaccgttg
    17281 ccctttagct gggacatcct gtctccacag tttatgtacg gttcaagagc ttttactaag
    17341 caccccgctg acatcccgga ctactataaa cagtcattcc ccgaaggatt caaatgggag
    17401 agggtcatga atttcgagga cggaggtgct gttacagtta cgcaggatac ctcacttgag
    17461 gatggcacgc ttatatataa ggtgaagctc cgcggtacga actttccgcc ggacggcccc
    17521 gttatgcaga agaagacgat gggctgggaa gcctctacgg aacgcctgta tcctgaggac
    17581 ggtgttctga aaggggatat taaaatggcc cttcggttga aagatggggg ccgctatctt
    17641 gcggacttta agaccaccta caaagcaaaa aagcctgtcc aaatgcccgg tgcatataac
    17701 gtcgatagaa aacttgatat aacctcccat aatgaagatt acacagtagt cgagcagtac
    17761 gaacgaagcg aaggtcggca ctcaactgga ggtatggatg aattgtataa gtaggatatc
    17821 atgcctaatt ttgagtgtat tactttcgaa aatcaacctc tggattacaa aatttgtgaa
    17881 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
    17941 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
    18001 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
    18061 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
    18121 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
    18181 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
    18241 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
    18301 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
    18361 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
    18421 ctttgggccg cctccccgca tcgataccga gcgctggtcg acagatctac gggtggcatc
    18481 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
    18541 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
    18601 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
    18661 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
    18721 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
    18781 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
    18841 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
    18901 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttgtt taaacgcgat
    18961 cgctta
//
