LOCUS       sequence-403363-       18437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Frt-inducible CRISPRi by Zim3-dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 18437)
  TITLE     CRISPRi by Zim3-dCas9 for iPSC Neurodegenerative Disease Initiative 
            (iNDI)
REFERENCE   2  (bases 1 to 18437)
  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: iF65 PB-FrtStopFrt-Zim3-dCas9-mScarlet-hygro
FEATURES             Location/Qualifiers
     source          1..18437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        9..388
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..666
                     /label=chicken beta-actin promoter
     intron          667..1683
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1607..1629
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1691..1710
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1756..1789
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    1956..2090
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2210..2344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2464..2598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2627..2660
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             join(3084..3110,3111..3113,3114..5600,5601..5603,
                     5604..7187)
                     /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             7197..7223
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7242..7262
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7269..7289
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7305..7358
                     /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             7359..7385
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             7401..7427
                     /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    8175..8763
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8228..8248)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8646..8657)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    8795..9271
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(8994..9013)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   9716..9778
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9908..9925)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9908..9924)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9917..9939)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10066..10521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10153..10172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10363..10384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10547..10651
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10652..10720,10721..11512)
                     /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(10870..10889)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11683..12271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12172..12191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12425..12442
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12559..12580
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12595..12600,12601..12618,12619..12625)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12633..12649
                     /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     12638..12660
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12657..12673
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12657..12673
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   13164..13198
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    13714..13747
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    13748..13972
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(13980..15005)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     intron          15028..15966
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(15051..15071)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(15967..16178)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    16179..16212
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ggcgcgccga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
       61 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      121 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      181 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      241 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      301 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      361 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggg cgcgcgccag gsggggcggg gcggggcgag
      541 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
      601 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      661 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      721 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      781 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
      841 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
      901 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
      961 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     1021 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     1081 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     1141 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     1201 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     1261 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     1321 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     1381 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     1441 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     1501 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     1561 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     1621 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     1681 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaatggtac
     1741 cgttaacgcg atcgcgaagt tcctattctc tagaaagtat aggaacttcc cgcgatgaat
     1801 aaatgaaagc ttgcagatct gcgaccctag aggatctgcg accctagagg atcataatca
     1861 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     1921 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     1981 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2041 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgcgacccta
     2101 gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     2161 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     2221 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2281 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2341 gatctgcgac cctagaggat cataatcagc cataccacat ttgtagaggt tttacttgct
     2401 ttaaaaaacc tcccacacct ccccctgaac ctgaaactaa aaatgaatgc aattgttgtt
     2461 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2521 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2581 tcttatcatg tctggatctc catcaagctg atccggaacc cttaatgaag ttcctattct
     2641 ctagaaagta taggaacttc gtatactcgc gagccgccac catgaacaac tctcagggca
     2701 gagtgacctt cgaggacgtg accgtgaact tcacccaagg cgagtggcag agactgaacc
     2761 ccgagcagag aaacctgtac cgggacgtga tgctggaaaa ctacagcaac ctggtgtccg
     2821 tcggccaggg cgagacaaca aagcctgacg tgatcctgcg gctggaacag ggcaaagagc
     2881 cttggctgga agaggaagag gtgctcggat ctggcagagc cgagaagaat ggcgacatcg
     2941 gcggacagat ctggaagccc aaggacgtga aagagtctct gagtggcggc tcaagtggcg
     3001 ggtctagtgg gagtgaaacg ccagggactt cagaatcagc aaccccggaa tcatccggcg
     3061 gatcatccgg tgggtctgta tacatggaca agaagtactc tatcggcctg gccatcggca
     3121 ccaattctgt tggatgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     3181 aggtgctggg caacaccgac cggcacagca tcaagaagaa tctgatcggc gccctgctgt
     3241 tcgactctgg cgaaacagcc gaagccacca gactgaagag aaccgccaga cggcggtaca
     3301 ccagaagaaa gaaccggatc tgctacctgc aagagatctt cagcaacgag atggccaagg
     3361 tggacgacag cttcttccac agactggaag agtccttcct ggtcgaggaa gataagaagc
     3421 acgagcggca ccccatcttc ggcaacatcg tggatgaggt ggcctaccac gagaagtacc
     3481 ctaccatcta ccacctccgg aagaaactgg tggacagcac cgacaaggcc gacctgagac
     3541 tgatctatct ggccctggct cacatgatca agttccgggg ccactttctg atcgagggcg
     3601 acctgaatcc tgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     3661 accagctgtt cgaggaaaac cccatcaacg ccagcggagt ggatgccaag gccatcctgt
     3721 ctgccagact gagcaagagc agacgcctgg aaaacctgat cgctcagctg cccggcgaga
     3781 agaagaacgg cctgttcgga aacctgattg ccctgagcct gggcctgaca cctaacttca
     3841 agagcaactt cgacctggct gaggacgcca aactgcagct gtccaaggac acctacgacg
     3901 acgacctgga caatctgctg gcccagatcg gcgatcagta cgccgacttg tttctggccg
     3961 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     4021 caaaggcccc tctgagcgcc tctatgatca agagatacga cgagcaccac caggatctga
     4081 ccctgctgaa ggccctcgtt agacagcagc tgcctgagaa gtacaaagag attttcttcg
     4141 accagagcaa gaacggctac gccggctaca ttgatggcgg agccagccaa gaggaatttt
     4201 acaagttcat caagcccatc cttgagaaga tggacggcac cgaggaactg ctggtcaagc
     4261 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caatggctct atccctcacc
     4321 agatccacct gggagagctg cacgccattc tgcggagaca agaggacttt tacccattcc
     4381 tgaaggacaa ccgggaaaag attgagaaga tcctgacctt cagaatcccc tactacgtgg
     4441 gaccactggc cagaggcaat agcagattcg cctggatgac cagaaagagc gaggaaacca
     4501 tcacaccctg gaacttcgag gaagtggtgg acaagggcgc cagcgctcag tccttcatcg
     4561 agcggatgac caacttcgat aagaacctgc ctaacgagaa ggtgctgccc aagcacagcc
     4621 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     4681 gaatgagaaa gcccgccttt ctgagcggcg agcagaaaaa ggccattgtg gatctgctgt
     4741 tcaagaccaa ccggaaagtg acagtgaagc agctgaaaga ggactacttc aagaaaatcg
     4801 agtgcttcga cagcgtggaa atcagcggcg tggaagatcg gttcaatgcc agcctgggca
     4861 cataccacga cctgctgaaa attatcaagg acaaggactt cctggacaac gaagagaacg
     4921 aggacatcct ggaagacatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     4981 aggaacggct gaaaacatac gcccacctgt tcgacgacaa agtgatgaag caactgaagc
     5041 ggcggagata caccggctgg ggtagactgt cccggaagct gatcaacggc atccgggata
     5101 agcagtccgg caagaccatc ctggactttc tgaagtccga cggcttcgcc aacagaaact
     5161 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
     5221 tgtccggcca aggcgattct ctgcacgagc acattgccaa cctggccgga tctcccgcca
     5281 ttaagaaggg catcctgcag accgtgaagg tggtggacga gcttgtgaaa gtgatgggca
     5341 gacacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acacagaagg
     5401 gccagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
     5461 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
     5521 tgtactacct gcagaatgga cgggatatgt acgtggacca agagctggac atcaaccggc
     5581 tgtccgacta cgatgtggac gctatcgtgc cccagtcttt tctgaaggac gactccatcg
     5641 acaacaaggt cctgaccaga agcgacaaga accggggcaa gagcgataac gtgccctccg
     5701 aagaggtggt caagaagatg aagaactatt ggcgccagct gctgaatgcc aagctgatta
     5761 cccagcggaa gttcgacaac ctgaccaagg ccgaaagagg cggcctgagc gaactggata
     5821 aggccggctt catcaagaga cagctggtgg aaacccggca gatcaccaaa cacgtggcac
     5881 agattctgga ctcccggatg aacactaagt acgacgagaa cgacaaactg atccgggaag
     5941 tgaaagtcat caccctgaag tccaagctgg tgtctgattt ccggaaggat ttccagttct
     6001 acaaagtgcg cgagatcaac aactaccatc acgcccacga cgcctacctg aatgccgttg
     6061 ttggaacagc cctgatcaag aagtatccca agctggaaag cgagttcgtg tacggcgact
     6121 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaagagatt ggcaaggcta
     6181 ccgccaagta ctttttctac tccaacatca tgaacttttt caagacagag atcaccctgg
     6241 ccaacggcga gatccggaaa agacccctga tcgagacaaa cggcgaaacc ggggagatcg
     6301 tgtgggataa gggcagagat tttgccacag tgcggaaagt gctgagcatg ccccaagtga
     6361 atatcgtgaa gaaaaccgag gtgcagacag gcggcttcag caaagagtct atcctgccta
     6421 agcggaacag cgacaagctg atcgcccgga agaaggactg ggacccaaag aagtacggcg
     6481 gcttcgatag ccctaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
     6541 agtccaagaa actcaagagc gtgaaagaac tgctcggcat caccatcatg gaacggtcca
     6601 gctttgagaa gaaccctatt gatttccttg aggccaaggg ctacaaagaa gtgaaaaagg
     6661 acctgatcat caagctcccc aagtactccc tgttcgagct ggaaaatggc cggaagcgga
     6721 tgctggcttc tgctggcgaa ctgcagaagg gaaacgaact ggccctgcct agcaaatatg
     6781 tgaactttct gtacctggcc agccactatg agaagctgaa gggcagcccc gaggacaatg
     6841 agcaaaagca gctgtttgtc gagcagcaca agcactacct ggacgagatc atcgagcaga
     6901 tcagcgagtt ctccaagaga gtgatcctgg ctgacgccaa tctggacaaa gtgctgtccg
     6961 cctacaacaa gcaccgggac aagcctatca gagagcaggc cgagaatatc atccacctgt
     7021 ttaccctgac caacctggga gcccctgccg ccttcaagta cttcgacacc accatcgacc
     7081 ggaagcgcta caccagcacc aaagaggttc tggacgccac actgatccac cagagcatca
     7141 ccggcctgta cgagacaaga atcgacctgt ctcagctcgg cggcgatcct cgaggctatc
     7201 cctatgacgt gcccgattat gccagcagtg gcagcggctc ccccaagaaa aaacgcaagg
     7261 tggaagatcc taagaaaaag cggaaagtgg ttaacggaag tggcgagggc agaggaagtc
     7321 tgctaacatg cggtgacgtc gaggagaatc ctggcccacc tgccgctaaa cgggtcaaac
     7381 tcgacggcgg ttctggaggt cctgctgcaa agcgcgttaa gttggatgct agcatggaat
     7441 ttgtgtctaa aggggaagca gtcattaaag agttcatgcg cttcaaagta cacatggaag
     7501 gaagtatgaa cgggcacgaa tttgaaatag aaggcgaagg ggaaggtcgg ccatacgaag
     7561 ggacgcaaac cgcgaagttg aaggttacaa agggaggacc gttgcccttt agctgggaca
     7621 tcctgtctcc acagtttatg tacggttcaa gagcttttac taagcacccc gctgacatcc
     7681 cggactacta taaacagtca ttccccgaag gattcaaatg ggagagggtc atgaatttcg
     7741 aggacggagg tgctgttaca gttacgcagg atacctcact tgaggatggc acgcttatat
     7801 ataaggtgaa gctccgcggt acgaactttc cgccggacgg ccccgttatg cagaagaaga
     7861 cgatgggctg ggaagcctct acggaacgcc tgtatcctga ggacggtgtt ctgaaagggg
     7921 atattaaaat ggcccttcgg ttgaaagatg ggggccgcta tcttgcggac tttaagacca
     7981 cctacaaagc aaaaaagcct gtccaaatgc ccggtgcata taacgtcgat agaaaacttg
     8041 atataacctc ccataatgaa gattacacag tagtcgagca gtacgaacga agcgaaggtc
     8101 ggcactcaac tggaggtatg gatgaattgt ataagtagga tatcatgcct aattttgagt
     8161 gtattacttt cgaaaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     8221 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     8281 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     8341 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     8401 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     8461 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     8521 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     8581 ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     8641 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     8701 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     8761 cgcatcgata ccgagcgctg gtcgacagat ctacgggtgg catccctgtg acccctcccc
     8821 agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa
     8881 attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg
     8941 ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg
     9001 ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt
     9061 tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag
     9121 gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt
     9181 ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca
     9241 ggcgtgaacc actgctccct tccctgtcct tgtttaaacg cgatcgctta attaaggccg
     9301 tggaggcctt ttccccgtat ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg
     9361 ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc
     9421 ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc
     9481 cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg gctgtccctc
     9541 ataaaagttt tgttacttta tagaagaaat tttgagtttt tgtttttttt aataaataaa
     9601 taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa
     9661 acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc
     9721 gtcaatttta cacatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt
     9781 aatctagctg cgtgttctgc agcgtgtcga gcatcttcat ctgctccatc acgctgtaaa
     9841 acacatttgc accgcgagtc tgcccgtcct ccacgggttc aaaaacgtga atgaacgagg
     9901 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     9961 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
    10021 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg
    10081 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
    10141 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
    10201 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
    10261 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
    10321 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
    10381 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
    10441 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
    10501 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
    10561 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
    10621 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
    10681 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
    10741 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
    10801 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
    10861 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
    10921 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
    10981 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
    11041 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
    11101 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
    11161 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
    11221 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
    11281 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
    11341 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
    11401 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
    11461 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
    11521 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
    11581 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
    11641 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
    11701 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
    11761 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
    11821 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
    11881 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
    11941 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
    12001 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
    12061 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
    12121 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
    12181 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
    12241 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
    12301 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
    12361 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
    12421 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
    12481 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
    12541 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
    12601 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
    12661 aaacagctat gaccatgatt acgccaagcg cgcccgccgg gtaactcacg gggtatccat
    12721 gtccatttct gcggcatcca gccaggatac ccgtcctcgc tgacgtaata tcccagcgcc
    12781 gcaccgctgt cattaatctg cacaccggca cggcagttcc ggctgtcgcc ggtattgttc
    12841 gggttgctga tgcgcttcgg gctgaccatc cggaactgtg tccggaaaag ccgcgacgaa
    12901 ctggtatccc aggtggcctg aacgaacagt tcaccgttaa aggcgtgcat ggccacacct
    12961 tcccgaatca tcatggtaaa cgtgcgtttt cgctcaacgt caatgcagca gcagtcatcc
    13021 tcggcaaact ctttccatgc cgcttcaacc tcgcgggaaa aggcacgggc ttcttcctcc
    13081 ccgatgccca gatagcgcca gcttgggcga tgactgagcc ggaaaaaaga cccgacgata
    13141 tgatcctgat gcagctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca
    13201 ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac
    13261 atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg
    13321 attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt
    13381 ttaagattta actcatacga taattatatt gttatttcat gttctactta cgtgataact
    13441 tattatatat atattttctt gttatagaat cagggacagc ccccccccaa agcccccagg
    13501 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
    13561 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
    13621 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
    13681 acctgggggg atacggggaa aaagccatac caaataactt cgtataatgt atgctatacg
    13741 aagttatcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc
    13801 cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc
    13861 gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag
    13921 gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac agcggccgcc
    13981 tattccttgg ctctgggtct tgtggagggt ctcctgtttc cagagtcagc cagaacttca
    14041 acacatccat cagtccaaac agcagcagac cttcttgcaa tttgagttct tccaacagtt
    14101 ccagcaccag acctcacaat ggcatcacat cttccttgtg cccaagcagc atcatcaaag
    14161 tttccatcaa ccagagattg atacagttgg tccaggccaa ttctgagcat gtaggctctc
    14221 agtctggggg aaccagccag ttctgggtgt cttctttcaa aatatcttgt ttgttgttcc
    14281 atgcaagcca gccaaggtct ccaaaaaaaa atgttggcaa cctcatattg agaatctcca
    14341 aacatggctt cagaccagtc aatgactgca gtgattctgc cattgtctgt cagaacattg
    14401 ttgcttccaa aatcagcatg gacaaggtgt ctgacttcag gacaatcttc tgcccacagc
    14461 atgagttcat ccagtgcctg agcaacagaa gcagaaactg tgtcatccat cacagtctgc
    14521 cagtgataga catgaggatc agcaatggca caaatgaaat ccctccaagt ggtgtactga
    14581 ccgatgcctt ggggaccaaa aggaccaaat ccgctggttt ggctcagatc agctgctgca
    14641 atggcatcca ttgcttcagc aacaggttgc agaacagctg gcagctcagt ttcagggaga
    14701 tcctggagag tgactccttg tgctcttctg ctgatgcagt aggtgagaga ttcagaaaat
    14761 tctccaatgt ccagaacttc tggaattggc agagcagcag aggcaaagtg tctgtaaaca
    14821 tatctgtctt tgtaaaaacc atcagcacaa gaattgaccc tcagaacata acctcttcct
    14881 ccaacatcaa aagaaaaggc tctgctttct tcaccttcag acagctgcat gagatcagaa
    14941 acagaatcaa atttttcaat gagaaacttc tcaacagaag ttgctgtcag ttcaggtttc
    15001 ttcatggtgg gatttaaatc acgacacctg aaatggaaga aaaaaacttt gaaccactgt
    15061 ctgaggcttg agaatgaacc aagatccaaa ctcaaaaagg gcaaattcca aggagaatta
    15121 catcaagtgc caagctggcc taacttcagt ctccacccac tcagtgtggg gaaactccat
    15181 cgcataaaac ccctcccccc aacctaaaga cgacgtactc caaaagcgcg agaactaatc
    15241 gaggtgcctg gacggcgccc ggtactccgt ggagtcacat gaagcgacgg ctgaggacgg
    15301 aaaggccctt ttcctttgtg tgggtgactc acccgcccgc tctcccgagc gccgcgtcct
    15361 ccattttgag ctccctgcag cagggccggg aagcggccat ctttccgctc acgcaactgg
    15421 tgccgaccgg gccagccttg ccgcccaggg cggggcgata cacggcggcg cgaggccagg
    15481 caccagagca ggccggccag cttgagacta cccccgtccg attctcggtg gccgcgctcg
    15541 caggccccgc ctcgccgaac atgtgcgctg ggacgcacgg gccccgtcgc cgcccgcggc
    15601 cccaaaaacc gaaataccag tgtgcagatc ttggcccgca tttacaagac tatcttgcca
    15661 gaaaaaaagc gtcgcagcag gtcatcaaaa attttaaatg gctagagact tatcgaaagc
    15721 agcgagacag gcgcgaaggt gccaccagat tcgcacgcgg cggccccagc gcccaggcca
    15781 ggcctcaact caagcacgag gcgaaggggc tccttaagcg caaggcctcg aactctccca
    15841 cccacttcca acccgaagct cgggatcaag aatcacgtac tgcagccagg tggaagtaat
    15901 tcaaggcacg caagggccat aacccgtaaa gaggccaggc ccgcgggaac cacacacggc
    15961 acttacctgt gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc
    16021 acttatatac ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact
    16081 ttcccagttt accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc
    16141 caacttctcg gggactgtgg gcgatgtgcg ctctgcccat aacttcgtat aatgtatgct
    16201 atacgaagtt ataatattct cactgactcc gtcctggagt tggatgagag ataatggcct
    16261 tacgttgtgc caggggaggg tcgggctgga tttagcaaga tttaccttct ccaaagagcg
    16321 gtgctgcagt ggcacagctg cccacggagg tgggggggtc accgtccctg gaggtgatga
    16381 agaactgtgg ggatgtggca ctgagggaca tggccagtgg gcacggtggg tgggttgggg
    16441 ttggtcttgg ggatcttgga gggcttttcc agccttcatg atttgacgat tgtatgaaca
    16501 tctacatggc aattctccag ctgcctgtcc cagtcctact gacccagctg tatctctcca
    16561 ggcaagctct tccacccctt ctgcttgcat ccagacacca tcaaacatgc aggctcagac
    16621 acagggacca gcagtgtctg tggccttttt gtgctcctct ccatgctggg ttttaacttg
    16681 ctctttgtcc ttctatccta tcttcttatc cttaaggctg ttctgaacgc tgtgacttgg
    16741 agagtgtccc agagccctca acacctgcat gtcccacgtc catgctgtcc tgcacttcct
    16801 tatccccaag atctgcctct ccgtgatgca ctgaattggc aaacatgtgt caccccagac
    16861 caacaatgtc acagcaaact cccccttgat aggacaaggg ggaatggctt tacactgaga
    16921 caggggaggt ttgggttgga tatgaggagg cagtttttcc cccagagggt ggtgacgcac
    16981 tgaacaggtt gcccaaggag gctgtggatg ccccatccct gcaggcattc aaggccaggc
    17041 tggatgtggc tctgggcagc ctgggctgct ggttgatgac cctgcacata gcagggggtt
    17101 ggatctggat gagcactgtg ctcctttgca acccaggccg ttctatgatt ctgtcattct
    17161 aaatctctct ttcagcctaa agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa
    17221 agagcagcga gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct
    17281 gtccccgcac gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg
    17341 ggcggctcgc tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg
    17401 gggggggctg tccccgtgag ctccacgtgc ctagggcaca cgaccacaat tccactgaaa
    17461 gcattttaat acggaacttg tcactcccag ggagcctccg ctcagccggc agttggttca
    17521 tttcaatccc cacgacaacc cttcaaagtg cagggcagac agcaggtggc tctgcccagg
    17581 cgcctggatc acagcccggc ctgcagccct cacctgggcg cggggagacc ctgaggacgc
    17641 tcctccaggc ggcgctggcc ggggcctgcg gacacggacg ggcgggctga gctccgggac
    17701 ccctccccgc gccccgcacc ccgcaccccg caccccgcac cccgcacccg gcgctcaccc
    17761 gtcccagccc cgccgcccgc agccccagct gcaacgcagc caccgccgcc atcgcacccg
    17821 gccccgcggg cgcttccggg acgcaggagg catctgcatc cggggcgccg ctgagtcccg
    17881 cccagagccc cgcccccggc tccaggttct gcgagcggct tccgccgggc tgctccgcgg
    17941 gcgcgtcggc catgagcgag ttgccgggcg acgtgcgggc gtttctgcgg gagcacccga
    18001 gcctgcggct ccagacggac gcccgcaagg ttcgcagcgc gggaggggaa cggagtggcg
    18061 gagaagggcg cagttgggat gaggggctga ggggagggca ggggagagga gagggcaggg
    18121 gagaggggag aggggagagc aggagagagg ggaaggcagg ggagagggcg cggcgggatc
    18181 aggggaggag agggaagggg gcgcggcagg agggggcacc agggagcgga gccctggccc
    18241 tcctgacgtc ctgcccgccc acgcgtccgc aggtgaggtg catcctgaca ggtcacgagc
    18301 tgccctgccg cctgccggag ctccaggtct acacccgcgg caaaaagtac cagcggctgg
    18361 tccgcgcctc cccggccttc gactatgcag agttcgagcc gcacatcgtg cccagcacca
    18421 agaacccgta ggtggtc
//
