LOCUS       sequence-403365-       18423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-inducible CRISPRi by Zim3-dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 18423)
  TITLE     CRISPRi by Zim3-dCas9 for iPSC Neurodegenerative Disease Initiative 
            (iNDI)
REFERENCE   2  (bases 1 to 18423)
  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: iF66 PB-LoxStopLox-Zim3-dCas9-mScarlet-hygro
FEATURES             Location/Qualifiers
     source          1..18423
                     /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    1742..1775
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     polyA_signal    1942..2076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2196..2330
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2450..2584
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2613..2646
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     CDS             join(3070..3096,3097..3099,3100..5586,5587..5589,
                     5590..7173)
                     /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             7183..7209
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7228..7248
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7255..7275
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7291..7344
                     /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             7345..7371
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             7387..7413
                     /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    8161..8749
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8214..8234)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8632..8643)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    8781..9257
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(8980..8999)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   9702..9764
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9894..9911)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9894..9910)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9903..9925)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10052..10507
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10139..10158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10349..10370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10533..10637
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10638..10706,10707..11498)
                     /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(10856..10875)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11669..12257
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12158..12177
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12411..12428
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12545..12566
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12581..12586,12587..12604,12605..12611)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12619..12635
                     /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     12624..12646
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12643..12659
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12643..12659
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   13150..13184
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    13700..13733
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    13734..13958
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(13966..14991)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     intron          15014..15952
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(15037..15057)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(15953..16164)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    16165..16198
                     /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 ggcgcgccag gsggggcggg gsggggcgag
      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 agaatgtcga
     1741 cataacttcg tataatgtat actatacgaa gttattcgcg atgaataaat gaaagcttgc
     1801 agatctgcga ccctagagga tctgcgaccc tagaggatca taatcagcca taccacattt
     1861 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     1921 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     1981 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2041 tccaaactca tcaatgtatc ttatcatgtc tggatctgcg accctagagg atcataatca
     2101 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     2161 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     2221 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2281 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgcgacccta
     2341 gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     2401 acacctcccc ctgaacctga aactaaaaat gaatgcaatt gttgttgtta acttgtttat
     2461 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2521 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2581 gatctccatc aagctgatcc ggaaccctta atataacttc gtataatgta tactatacga
     2641 agttatggta cctcgcgagc cgccaccatg aacaactctc agggcagagt gaccttcgag
     2701 gacgtgaccg tgaacttcac ccaaggcgag tggcagagac tgaaccccga gcagagaaac
     2761 ctgtaccggg acgtgatgct ggaaaactac agcaacctgg tgtccgtcgg ccagggcgag
     2821 acaacaaagc ctgacgtgat cctgcggctg gaacagggca aagagccttg gctggaagag
     2881 gaagaggtgc tcggatctgg cagagccgag aagaatggcg acatcggcgg acagatctgg
     2941 aagcccaagg acgtgaaaga gtctctgagt ggcggctcaa gtggcgggtc tagtgggagt
     3001 gaaacgccag ggacttcaga atcagcaacc ccggaatcat ccggcggatc atccggtggg
     3061 tctgtataca tggacaagaa gtactctatc ggcctggcca tcggcaccaa ttctgttgga
     3121 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     3181 accgaccggc acagcatcaa gaagaatctg atcggcgccc tgctgttcga ctctggcgaa
     3241 acagccgaag ccaccagact gaagagaacc gccagacggc ggtacaccag aagaaagaac
     3301 cggatctgct acctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     3361 ttccacagac tggaagagtc cttcctggtc gaggaagata agaagcacga gcggcacccc
     3421 atcttcggca acatcgtgga tgaggtggcc taccacgaga agtaccctac catctaccac
     3481 ctccggaaga aactggtgga cagcaccgac aaggccgacc tgagactgat ctatctggcc
     3541 ctggctcaca tgatcaagtt ccggggccac tttctgatcg agggcgacct gaatcctgac
     3601 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     3661 gaaaacccca tcaacgccag cggagtggat gccaaggcca tcctgtctgc cagactgagc
     3721 aagagcagac gcctggaaaa cctgatcgct cagctgcccg gcgagaagaa gaacggcctg
     3781 ttcggaaacc tgattgccct gagcctgggc ctgacaccta acttcaagag caacttcgac
     3841 ctggctgagg acgccaaact gcagctgtcc aaggacacct acgacgacga cctggacaat
     3901 ctgctggccc agatcggcga tcagtacgcc gacttgtttc tggccgccaa gaacctgtcc
     3961 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcacaaa ggcccctctg
     4021 agcgcctcta tgatcaagag atacgacgag caccaccagg atctgaccct gctgaaggcc
     4081 ctcgttagac agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     4141 ggctacgccg gctacattga tggcggagcc agccaagagg aattttacaa gttcatcaag
     4201 cccatccttg agaagatgga cggcaccgag gaactgctgg tcaagctgaa cagagaggac
     4261 ctgctgcgga agcagcggac cttcgacaat ggctctatcc ctcaccagat ccacctggga
     4321 gagctgcacg ccattctgcg gagacaagag gacttttacc cattcctgaa ggacaaccgg
     4381 gaaaagattg agaagatcct gaccttcaga atcccctact acgtgggacc actggccaga
     4441 ggcaatagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac accctggaac
     4501 ttcgaggaag tggtggacaa gggcgccagc gctcagtcct tcatcgagcg gatgaccaac
     4561 ttcgataaga acctgcctaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     4621 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     4681 gcctttctga gcggcgagca gaaaaaggcc attgtggatc tgctgttcaa gaccaaccgg
     4741 aaagtgacag tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgacagc
     4801 gtggaaatca gcggcgtgga agatcggttc aatgccagcc tgggcacata ccacgacctg
     4861 ctgaaaatta tcaaggacaa ggacttcctg gacaacgaag agaacgagga catcctggaa
     4921 gacatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     4981 acatacgccc acctgttcga cgacaaagtg atgaagcaac tgaagcggcg gagatacacc
     5041 ggctggggta gactgtcccg gaagctgatc aacggcatcc gggataagca gtccggcaag
     5101 accatcctgg actttctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     5161 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccaaggc
     5221 gattctctgc acgagcacat tgccaacctg gccggatctc ccgccattaa gaagggcatc
     5281 ctgcagaccg tgaaggtggt ggacgagctt gtgaaagtga tgggcagaca caagcccgag
     5341 aacatcgtga tcgaaatggc cagagagaac cagaccacac agaagggcca gaagaacagc
     5401 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     5461 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     5521 aatggacggg atatgtacgt ggaccaagag ctggacatca accggctgtc cgactacgat
     5581 gtggacgcta tcgtgcccca gtcttttctg aaggacgact ccatcgacaa caaggtcctg
     5641 accagaagcg acaagaaccg gggcaagagc gataacgtgc cctccgaaga ggtggtcaag
     5701 aagatgaaga actattggcg ccagctgctg aatgccaagc tgattaccca gcggaagttc
     5761 gacaacctga ccaaggccga aagaggcggc ctgagcgaac tggataaggc cggcttcatc
     5821 aagagacagc tggtggaaac ccggcagatc accaaacacg tggcacagat tctggactcc
     5881 cggatgaaca ctaagtacga cgagaacgac aaactgatcc gggaagtgaa agtcatcacc
     5941 ctgaagtcca agctggtgtc tgatttccgg aaggatttcc agttctacaa agtgcgcgag
     6001 atcaacaact accatcacgc ccacgacgcc tacctgaatg ccgttgttgg aacagccctg
     6061 atcaagaagt atcccaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     6121 gtgcggaaga tgatcgccaa gagcgagcaa gagattggca aggctaccgc caagtacttt
     6181 ttctactcca acatcatgaa ctttttcaag acagagatca ccctggccaa cggcgagatc
     6241 cggaaaagac ccctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     6301 agagattttg ccacagtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaagaaa
     6361 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcctaagcg gaacagcgac
     6421 aagctgatcg cccggaagaa ggactgggac ccaaagaagt acggcggctt cgatagccct
     6481 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactc
     6541 aagagcgtga aagaactgct cggcatcacc atcatggaac ggtccagctt tgagaagaac
     6601 cctattgatt tccttgaggc caagggctac aaagaagtga aaaaggacct gatcatcaag
     6661 ctccccaagt actccctgtt cgagctggaa aatggccgga agcggatgct ggcttctgct
     6721 ggcgaactgc agaagggaaa cgaactggcc ctgcctagca aatatgtgaa ctttctgtac
     6781 ctggccagcc actatgagaa gctgaagggc agccccgagg acaatgagca aaagcagctg
     6841 tttgtcgagc agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     6901 aagagagtga tcctggctga cgccaatctg gacaaagtgc tgtccgccta caacaagcac
     6961 cgggacaagc ctatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaac
     7021 ctgggagccc ctgccgcctt caagtacttc gacaccacca tcgaccggaa gcgctacacc
     7081 agcaccaaag aggttctgga cgccacactg atccaccaga gcatcaccgg cctgtacgag
     7141 acaagaatcg acctgtctca gctcggcggc gatcctcgag gctatcccta tgacgtgccc
     7201 gattatgcca gcagtggcag cggctccccc aagaaaaaac gcaaggtgga agatcctaag
     7261 aaaaagcgga aagtggttaa cggaagtggc gagggcagag gaagtctgct aacatgcggt
     7321 gacgtcgagg agaatcctgg cccacctgcc gctaaacggg tcaaactcga cggcggttct
     7381 ggaggtcctg ctgcaaagcg cgttaagttg gatgctagca tggaatttgt gtctaaaggg
     7441 gaagcagtca ttaaagagtt catgcgcttc aaagtacaca tggaaggaag tatgaacggg
     7501 cacgaatttg aaatagaagg cgaaggggaa ggtcggccat acgaagggac gcaaaccgcg
     7561 aagttgaagg ttacaaaggg aggaccgttg ccctttagct gggacatcct gtctccacag
     7621 tttatgtacg gttcaagagc ttttactaag caccccgctg acatcccgga ctactataaa
     7681 cagtcattcc ccgaaggatt caaatgggag agggtcatga atttcgagga cggaggtgct
     7741 gttacagtta cgcaggatac ctcacttgag gatggcacgc ttatatataa ggtgaagctc
     7801 cgcggtacga actttccgcc ggacggcccc gttatgcaga agaagacgat gggctgggaa
     7861 gcctctacgg aacgcctgta tcctgaggac ggtgttctga aaggggatat taaaatggcc
     7921 cttcggttga aagatggggg ccgctatctt gcggacttta agaccaccta caaagcaaaa
     7981 aagcctgtcc aaatgcccgg tgcatataac gtcgatagaa aacttgatat aacctcccat
     8041 aatgaagatt acacagtagt cgagcagtac gaacgaagcg aaggtcggca ctcaactgga
     8101 ggtatggatg aattgtataa gtaggatatc atgcctaatt ttgagtgtat tactttcgaa
     8161 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     8221 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     8281 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     8341 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     8401 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     8461 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     8521 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     8581 gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     8641 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     8701 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga
     8761 gcgctggtcg acagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     8821 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     8881 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     8941 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     9001 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     9061 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     9121 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
     9181 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     9241 ctcccttccc tgtccttgtt taaacgcgat cgcttaatta aggccgtgga ggccttttcc
     9301 ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg
     9361 atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg
     9421 gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga
     9481 gggacgtaat tacatccctg ggggctttgg gggggggctg tccctcataa aagttttgtt
     9541 actttataga agaaattttg agtttttgtt ttttttaata aataaataaa cataaataaa
     9601 ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt aatatctatt
     9661 caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca attttacaca
     9721 tgattatctt taacgtacgt cacaatatga ttatctttct agggttaatc tagctgcgtg
     9781 ttctgcagcg tgtcgagcat cttcatctgc tccatcacgc tgtaaaacac atttgcaccg
     9841 cgagtctgcc cgtcctccac gggttcaaaa acgtgaatga acgaggcgcg ctcactggcc
     9901 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     9961 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
    10021 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
    10081 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
    10141 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
    10201 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
    10261 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
    10321 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
    10381 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
    10441 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
    10501 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
    10561 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
    10621 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
    10681 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
    10741 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
    10801 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
    10861 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
    10921 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
    10981 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
    11041 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
    11101 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
    11161 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
    11221 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
    11281 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
    11341 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
    11401 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
    11461 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
    11521 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
    11581 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    11641 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    11701 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    11761 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
    11821 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    11881 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    11941 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    12001 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    12061 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    12121 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
    12181 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
    12241 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
    12301 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
    12361 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
    12421 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
    12481 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
    12541 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
    12601 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
    12661 atgattacgc caagcgcgcc cgccgggtaa ctcacggggt atccatgtcc atttctgcgg
    12721 catccagcca ggatacccgt cctcgctgac gtaatatccc agcgccgcac cgctgtcatt
    12781 aatctgcaca ccggcacggc agttccggct gtcgccggta ttgttcgggt tgctgatgcg
    12841 cttcgggctg accatccgga actgtgtccg gaaaagccgc gacgaactgg tatcccaggt
    12901 ggcctgaacg aacagttcac cgttaaaggc gtgcatggcc acaccttccc gaatcatcat
    12961 ggtaaacgtg cgttttcgct caacgtcaat gcagcagcag tcatcctcgg caaactcttt
    13021 ccatgccgct tcaacctcgc gggaaaaggc acgggcttct tcctccccga tgcccagata
    13081 gcgccagctt gggcgatgac tgagccggaa aaaagacccg acgatatgat cctgatgcag
    13141 ctagattaac cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg
    13201 ctctctcttt ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc
    13261 ttggagctcc cgtgaggcgt gcttgtcaat gcggtaagtg tcactgattt tgaactataa
    13321 cgaccgcgtg agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc
    13381 atacgataat tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat
    13441 tttcttgtta tagaatcagg gacagccccc ccccaaagcc cccagggatg taattacgtc
    13501 cctcccccgc tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc
    13561 cccgcatccc cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg
    13621 atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct ggggggatac
    13681 ggggaaaaag ccataccaaa taacttcgta taatgtatgc tatacgaagt tatccataga
    13741 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
    13801 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
    13861 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
    13921 ggggcaaaca acagatggct ggcaactaga aggcacagcg gccgcctatt ccttggctct
    13981 gggtcttgtg gagggtctcc tgtttccaga gtcagccaga acttcaacac atccatcagt
    14041 ccaaacagca gcagaccttc ttgcaatttg agttcttcca acagttccag caccagacct
    14101 cacaatggca tcacatcttc cttgtgccca agcagcatca tcaaagtttc catcaaccag
    14161 agattgatac agttggtcca ggccaattct gagcatgtag gctctcagtc tgggggaacc
    14221 agccagttct gggtgtcttc tttcaaaata tcttgtttgt tgttccatgc aagccagcca
    14281 aggtctccaa aaaaaaatgt tggcaacctc atattgagaa tctccaaaca tggcttcaga
    14341 ccagtcaatg actgcagtga ttctgccatt gtctgtcaga acattgttgc ttccaaaatc
    14401 agcatggaca aggtgtctga cttcaggaca atcttctgcc cacagcatga gttcatccag
    14461 tgcctgagca acagaagcag aaactgtgtc atccatcaca gtctgccagt gatagacatg
    14521 aggatcagca atggcacaaa tgaaatccct ccaagtggtg tactgaccga tgccttgggg
    14581 accaaaagga ccaaatccgc tggtttggct cagatcagct gctgcaatgg catccattgc
    14641 ttcagcaaca ggttgcagaa cagctggcag ctcagtttca gggagatcct ggagagtgac
    14701 tccttgtgct cttctgctga tgcagtaggt gagagattca gaaaattctc caatgtccag
    14761 aacttctgga attggcagag cagcagaggc aaagtgtctg taaacatatc tgtctttgta
    14821 aaaaccatca gcacaagaat tgaccctcag aacataacct cttcctccaa catcaaaaga
    14881 aaaggctctg ctttcttcac cttcagacag ctgcatgaga tcagaaacag aatcaaattt
    14941 ttcaatgaga aacttctcaa cagaagttgc tgtcagttca ggtttcttca tggtgggatt
    15001 taaatcacga cacctgaaat ggaagaaaaa aactttgaac cactgtctga ggcttgagaa
    15061 tgaaccaaga tccaaactca aaaagggcaa attccaagga gaattacatc aagtgccaag
    15121 ctggcctaac ttcagtctcc acccactcag tgtggggaaa ctccatcgca taaaacccct
    15181 ccccccaacc taaagacgac gtactccaaa agcgcgagaa ctaatcgagg tgcctggacg
    15241 gcgcccggta ctccgtggag tcacatgaag cgacggctga ggacggaaag gcccttttcc
    15301 tttgtgtggg tgactcaccc gcccgctctc ccgagcgccg cgtcctccat tttgagctcc
    15361 ctgcagcagg gccgggaagc ggccatcttt ccgctcacgc aactggtgcc gaccgggcca
    15421 gccttgccgc ccagggcggg gcgatacacg gcggcgcgag gccaggcacc agagcaggcc
    15481 ggccagcttg agactacccc cgtccgattc tcggtggccg cgctcgcagg ccccgcctcg
    15541 ccgaacatgt gcgctgggac gcacgggccc cgtcgccgcc cgcggcccca aaaaccgaaa
    15601 taccagtgtg cagatcttgg cccgcattta caagactatc ttgccagaaa aaaagcgtcg
    15661 cagcaggtca tcaaaaattt taaatggcta gagacttatc gaaagcagcg agacaggcgc
    15721 gaaggtgcca ccagattcgc acgcggcggc cccagcgccc aggccaggcc tcaactcaag
    15781 cacgaggcga aggggctcct taagcgcaag gcctcgaact ctcccaccca cttccaaccc
    15841 gaagctcggg atcaagaatc acgtactgca gccaggtgga agtaattcaa ggcacgcaag
    15901 ggccataacc cgtaaagagg ccaggcccgc gggaaccaca cacggcactt acctgtgttc
    15961 tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac tactgcactt atatacggtt
    16021 ctcccccacc ctcgggaaaa aggcggagcc agtacacgac atcactttcc cagtttaccc
    16081 cgcgccacct tctctaggca ccggttcaat tgccgacccc tccccccaac ttctcgggga
    16141 ctgtgggcga tgtgcgctct gcccataact tcgtataatg tatgctatac gaagttataa
    16201 tattctcact gactccgtcc tggagttgga tgagagataa tggccttacg ttgtgccagg
    16261 ggagggtcgg gctggattta gcaagattta ccttctccaa agagcggtgc tgcagtggca
    16321 cagctgccca cggaggtggg ggggtcaccg tccctggagg tgatgaagaa ctgtggggat
    16381 gtggcactga gggacatggc cagtgggcac ggtgggtggg ttggggttgg tcttggggat
    16441 cttggagggc ttttccagcc ttcatgattt gacgattgta tgaacatcta catggcaatt
    16501 ctccagctgc ctgtcccagt cctactgacc cagctgtatc tctccaggca agctcttcca
    16561 ccccttctgc ttgcatccag acaccatcaa acatgcaggc tcagacacag ggaccagcag
    16621 tgtctgtggc ctttttgtgc tcctctccat gctgggtttt aacttgctct ttgtccttct
    16681 atcctatctt cttatcctta aggctgttct gaacgctgtg acttggagag tgtcccagag
    16741 ccctcaacac ctgcatgtcc cacgtccatg ctgtcctgca cttccttatc cccaagatct
    16801 gcctctccgt gatgcactga attggcaaac atgtgtcacc ccagaccaac aatgtcacag
    16861 caaactcccc cttgatagga caagggggaa tggctttaca ctgagacagg ggaggtttgg
    16921 gttggatatg aggaggcagt ttttccccca gagggtggtg acgcactgaa caggttgccc
    16981 aaggaggctg tggatgcccc atccctgcag gcattcaagg ccaggctgga tgtggctctg
    17041 ggcagcctgg gctgctggtt gatgaccctg cacatagcag ggggttggat ctggatgagc
    17101 actgtgctcc tttgcaaccc aggccgttct atgattctgt cattctaaat ctctctttca
    17161 gcctaaagct ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag
    17221 cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg
    17281 ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct
    17341 gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc
    17401 cgtgagctcc acgtgcctag ggcacacgac cacaattcca ctgaaagcat tttaatacgg
    17461 aacttgtcac tcccagggag cctccgctca gccggcagtt ggttcatttc aatccccacg
    17521 acaacccttc aaagtgcagg gcagacagca ggtggctctg cccaggcgcc tggatcacag
    17581 cccggcctgc agccctcacc tgggcgcggg gagaccctga ggacgctcct ccaggcggcg
    17641 ctggccgggg cctgcggaca cggacgggcg ggctgagctc cgggacccct ccccgcgccc
    17701 cgcaccccgc accccgcacc ccgcaccccg cacccggcgc tcacccgtcc cagccccgcc
    17761 gcccgcagcc ccagctgcaa cgcagccacc gccgccatcg cacccggccc cgcgggcgct
    17821 tccgggacgc aggaggcatc tgcatccggg gcgccgctga gtcccgccca gagccccgcc
    17881 cccggctcca ggttctgcga gcggcttccg ccgggctgct ccgcgggcgc gtcggccatg
    17941 agcgagttgc cgggcgacgt gcgggcgttt ctgcgggagc acccgagcct gcggctccag
    18001 acggacgccc gcaaggttcg cagcgcggga ggggaacgga gtggcggaga agggcgcagt
    18061 tgggatgagg ggctgagggg agggcagggg agaggagagg gcaggggaga ggggagaggg
    18121 gagagcagga gagaggggaa ggcaggggag agggcgcggc gggatcaggg gaggagaggg
    18181 aagggggcgc ggcaggaggg ggcaccaggg agcggagccc tggccctcct gacgtcctgc
    18241 ccgcccacgc gtccgcaggt gaggtgcatc ctgacaggtc acgagctgcc ctgccgcctg
    18301 ccggagctcc aggtctacac ccgcggcaaa aagtaccagc ggctggtccg cgcctccccg
    18361 gccttcgact atgcagagtt cgagccgcac atcgtgccca gcaccaagaa cccgtaggtg
    18421 gtc
//
