LOCUS       sequence-434915-       14057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline-inducible (Zim3)KRAB-dCas9-HA-P2A-mCherry cassette for 
            integration at the AAVS1-locus of the cynomolgus genome..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14057)
  AUTHORS   Edenhofer FC, Termeg A, Ohnuki M, Jocher J, Kliesmete Z, Briem E, 
            Hellmann I, Enard W
  TITLE     Generation and characterization of inducible KRAB-dCas9 iPSCs from 
            primates for cross-species CRISPRi.
  JOURNAL   iScience. 2024 May 23;27(6):110090. doi: 10.1016/j.isci.2024.110090.
            eCollection 2024 Jun 21.
  PUBMED    38947524
REFERENCE   2  (bases 1 to 14057)
  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"; journalName: "iScience.";
            date: "2024-05-23"; volume: "27(6):110090. doi"; pages: " 
            10.1016/j.isci.2024.110090. eCollection 2024 Jun 21"
COMMENT     Sequence Label: pCyno-AAVS1-TetOn-ZIM3-KRAB-dCas9-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..14057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(844..912,913..1704)
                     /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(1062..1081)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1875..2463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2364..2383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3388..3406
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3424..3442
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3460..3478
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3496..3514
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3532..3550
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3568..3586
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3604..3622
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3667..3691
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3773..3797
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4146..4170)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4190..4210
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4259..4268
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4265..4291,4292..4294,4295..6781,6782..6784,
                     6785..8368)
                     /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             4265..8368
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /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             8372..8398
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             8378..8404
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             8417..8437
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8444..8464
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8483..8539
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             8540..9250
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(8685..8703)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     8953..8972
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    9282..9403
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9319..9338)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9373..9392
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9452..9468)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9452..9468)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9465..9487)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9476..9492
                     /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)."
     promoter        complement(join(9500..9506,9507..9524,9525..9530))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9545..9566
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9683..9700)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9756..9789
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     10120..10139
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    10138..10193
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     10239..10258
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(10345..11091)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(11123..11142)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          11150..12166
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(11204..11226)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        12446..12825
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    12831..13055
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     13044..13061
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(13081..13884)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(13192..13211)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     13802..13821
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     regulatory      13881..13890
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(13906..13959)
                     /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"
     misc_feature    13983..14008
                     /label=SA
                     /note="splice acceptor site"
ORIGIN
        1 gccccacggc ggggtggagg ggacagataa aagtacccag aaccagagcc atattaatcg
       61 gtcctgggaa tataaggtgg tcccggctct gggacacaag atcttgtcct gaagtggcca
      121 tgggggcccg gcttggaaga agaggaccag ccgagctctg ggacccctgg aagatgccgt
      181 gacaggggcc ggaagagcga gcacagacta gagaggtaaa gggggtatgg gagctgcccg
      241 aattaaagaa gtgagaggtg acccaaatcc acaggaggag ggggtgtcca ggcaaagaaa
      301 gtaagaggat ggagaggtgg ctaaagccag ggagacaggg actttggggt tgtcgggaaa
      361 aacggtgatg atgcaggcct acgagaaggg gaggtgggga cgcaagggag aggcatccca
      421 cagagaagac catcctaaga aacgagagat ggcacaggcc ccagaaggag agggaaaagg
      481 gaacccagcg aatgaagatg gcatgggggt tggggtaggg agcagagatg cccggagagg
      541 acccagacaa gggcaggatc cgctccgagg acaccacgtg gtgcagcgcc gagaaggaag
      601 tgctctggaa agagcatcct tgggcagcaa cgcacagcac agagcaaggg gaagagggat
      661 tggaggaagc tggaacctga aggaggtggt ggggaaggat ctgggcctag ccgtaggggt
      721 ggcccaggcc acaagctgca ggcagaaagc ggcgcaggtt caggggagag aatgcaggtc
      781 agtgaaagca aagggcgaat tcaaccctga taaatgcttc aataatattg aaaaaggaag
      841 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
      901 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
      961 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     1021 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     1081 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     1141 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     1201 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     1261 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     1321 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     1381 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     1441 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     1501 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     1561 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     1621 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     1681 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     1741 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     1801 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1861 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1921 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1981 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     2041 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2101 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2161 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2221 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2281 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2341 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2401 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2461 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcga attcgccctt agagcagagc
     2521 caggaacccc tgtagggaaa gtgcaggaga gccaggggca tgggatggtg acgaggaagg
     2581 gggacaggga acctgagcgc ctctcctggg cttgccaagg actgaaaccc aggggcccgg
     2641 agcagggcct tagggaagag gggccccggg accccgctcc gggcggagga gtatgtccca
     2701 gatagcactg ggcctcttta aggaaagaag gatggagaaa gagaaggggg tagaggcggc
     2761 cacgacctgg tgaacaccta ggacgaacca ttctcacaaa gggagttttc cacacggaca
     2821 cgccctcctc gccacagccc tgccagggcg gggcggctac tggccttatc tcacagggaa
     2881 aactgatgca cagaggaaca gtacaaattc ggggctagaa aggtgaagac ccaaaattag
     2941 aactcaggac caacttattc tgattttgtt tttccaaact gcttctcctc ttgggaagcg
     3001 taaggaagca gcagcacccg gctcagtgaa atgcaccaga cggccgcgcc agagcagctc
     3061 aggctccggg agagggcagc gcagggcggc cactgagaac cagcaggtcg cgcatccccc
     3121 ttacctccca ccccccgcca agctctccct cccaggatcc tctctggctc catcagtaag
     3181 caaaccctag gggttctggc ggggagagag aggtggctcc aggaaatggg ggtgtgtcac
     3241 cagataagga atgtgcttaa aaggaggtgg gggtcaggcc caatatctgg agactaggag
     3301 ggaggaggcc taaggatggg gctttttgtc accagtcctg tcccagtaaa gctgatatct
     3361 ccaccccaca gtggcgcgcc ggtttactcc ctatcagtga tagagaacgt atgaagagtt
     3421 tactccctat cagtgataga gaacgtatgc agactttact ccctatcagt gatagagaac
     3481 gtataaggag tttactccct atcagtgata gagaacgtat gaccagttta ctccctatca
     3541 gtgatagaga acgtatctac agtttactcc ctatcagtga tagagaacgt atatccagtt
     3601 tactccctat cagtgataga gaacgtataa gctttaggcg tgtacggtgg gcgcctataa
     3661 aagcagagct cgtttagtga accgtcagat cgcctggagc aattccacaa cacttttgtc
     3721 ttataccaac tttccgtacc acttcctacc ctcgtaaact taaggttaat caacaagttt
     3781 gtacaaaaaa gcaggctggc gccggaacca attcagtcga caggcctagg ctacgcgcgc
     3841 caccatgaac aattcccagg gaagagtgac cttcgaggat gtcactgtga acttcaccca
     3901 gggggagtgg cagcggctga atcccgaaca gagaaacttg tacagggatg tgatgctgga
     3961 gaattacagc aaccttgtct ctgtgggaca aggggaaacc accaaacccg atgtgatctt
     4021 gaggttggaa caaggaaagg agccatggtt ggaggaagag gaagtgctgg gaagtggccg
     4081 tgcagaaaaa aatggggaca ttggagggca gatttggaag ccaaaggatg tgaaagagag
     4141 tctctaccca actttcttgt acaaagtggt tggtggatct ggaggttctc ccaagaagaa
     4201 gcgcaaggtg ggacgcgtct gcaggatatc aagcttgcgg taccgcgggc ccgggatcgc
     4261 caccatggac aagaagtaca gcatcggcct ggccatcggc accaactctg tgggctgggc
     4321 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     4381 ccggcacagc atcaagaaga acctgatcgg cgccctgctg ttcgacagcg gagaaacagc
     4441 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     4501 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     4561 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     4621 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     4681 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     4741 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     4801 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     4861 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     4921 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     4981 caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     5041 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     5101 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     5161 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     5221 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     5281 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     5341 cgccggctac atcgatggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     5401 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     5461 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     5521 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     5581 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     5641 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     5701 ggaagtggtg gacaagggcg ccagcgccca gagcttcatc gagcggatga ccaacttcga
     5761 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     5821 cgtgtacaac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     5881 cctgagcggc gagcagaaaa aagccatcgt ggacctgctg ttcaagacca accggaaagt
     5941 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     6001 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     6061 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     6121 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     6181 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     6241 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     6301 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     6361 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     6421 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     6481 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     6541 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     6601 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     6661 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     6721 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     6781 cgctatcgtg cctcagagct ttctgaagga cgactccatc gataacaaag tgctgactcg
     6841 gagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     6901 gaagaactac tggcgccagc tgctgaatgc caagctgatt acccagagga agttcgacaa
     6961 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     7021 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     7081 gaacactaag tacgacgaga acgacaaact gatccgggaa gtgaaagtga tcaccctgaa
     7141 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     7201 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     7261 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     7321 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     7381 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     7441 gcggcctctg atcgagacaa acggcgaaac aggcgagatc gtgtgggata agggccggga
     7501 ctttgccacc gtgcggaaag tgctgtctat gccccaagtg aatatcgtga aaaagaccga
     7561 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgacaagct
     7621 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     7681 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     7741 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     7801 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     7861 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     7921 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     7981 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     8041 ggaacagcac aaacactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     8101 agtgatcctg gccgacgcta atctggacaa ggtgctgagc gcctacaaca agcacagaga
     8161 caagcctatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     8221 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     8281 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     8341 gatcgacctg tctcagctgg gaggcgacgc ctatccctat gacgtgcccg attatgccag
     8401 cctgggcagc ggctccccca agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa
     8461 agtggacggc attggtagtg gggctactaa cttcagcctg ctgaagcagg ctggagacgt
     8521 ggaggagaac cctggaccta tggtgagcaa gggcgaggag gataacatgg ccatcatcaa
     8581 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
     8641 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     8701 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
     8761 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     8821 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     8881 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     8941 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
     9001 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
     9061 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
     9121 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
     9181 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
     9241 gtacaagtaa accggtgcgg ccgcaatcga tcgcccaccc caacttgttt attgcagctt
     9301 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     9361 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     9421 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     9481 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     9541 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     9601 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     9661 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     9721 ggcgagcggt atcagctcac tcaaaggacg gccgcataac ttcgtataat gtatgctata
     9781 cgaagttata agcttgggct gcaggtcgag ggatctccat aagagaagag ggacagctat
     9841 gactgggagt agtcaggaga ggaggaaaaa tctggctagt aaaacatgta aggaaaattt
     9901 tagggatgtt aaagaaaaaa ataacacaaa acaaaatata aaaaaaatct aacctcaagt
     9961 caaggctttt ctatggaata aggaatggac agcagggggc tgtttcatat actgatgacc
    10021 tctttatagc caacctttgt tcatggcagc cagcatatgg gcatatgttg ccaaactcta
    10081 aaccaaatac tcattctgat gttttaaatg atttgccctc ccatatgtcc ttccgagtga
    10141 gagacacaaa aaattccaac acactattgc aatgaaaata aatttccttt attagccaga
    10201 agtcagatgc tcaaggggct tcatgatgtc cccataattt ttggcagagg gaaaaagatc
    10261 tcagtggtat ttgtgagcca gggcattggc cacaccagcc accaccttct gataggcagc
    10321 ctgcacctga ggagtgaatt cattttagcc tggcagcata tcgagatcga agtcatcgag
    10381 agcgtcagca gggagcatat ccagatcaaa atcatccagg gcatcggcgg gcagcatgtc
    10441 caggtcgaag tcgtccaggg cgtcggtggg tccgccgctc tcgcacttca gctgcttctc
    10501 gaggccgcag atgatcagtt ccaggccgaa caggaaggcg ggctcggcgc cctgtctgtc
    10561 gaacagctcg atggcctgct tcagcagggg gggcatgctg tcggtggtgg gtgtctctct
    10621 ttcctctttg gcgacctggt gttcctgttc ttccagcacg cagcccaggg tgaagtggcc
    10681 cacggcgctc agggcgtaca gggcgttttc caggctgaag ccctgctggc acaggaaggc
    10741 cagctggttt tccagtgtct cgtactgctt ctcggtgggt ctggtgccca ggtgcacttt
    10801 ggcgccgtcc cggtggctca gcagggcgca tctgtagctc ttggcgttgt tccgcaggaa
    10861 gtcctgccag ctctcgcctt ccagagggca gctgtgggtg tggtgccggt ccagcatctc
    10921 gatgggcagg gcgtccagca gggcccgctt gttcttcacg tgccagtaca gggtgggctg
    10981 ttccacgccc agtttctggg ccagcttccg ggtggtcagg ccctcgatgc ccacgccgtt
    11041 cagcagttcc agggcgctgt tgatcacttt gctcttgtcc agccggctca tgtcgacatt
    11101 ctttgccaaa atgatgagac agcacaataa ccagcacgtt gcccaggagc tgtaggaaaa
    11161 agaagaaggc atgaacatgg ttagcagagg ctctagagcc gccggtcaca cgccagaagc
    11221 cgaaccccgc cctgccccgt cccccccgaa ggcagccgtc cccccgcgga cagccccgag
    11281 gctggagagg gagaagggga cggcggcgcg gcgacgcacg aaggccctcc ccgcccattt
    11341 ccttcctgcc ggcgccgcac cgcttcgccc cgcgcccgct agagggggtg cggcggcgcc
    11401 tcccagattt cggctccgca cagatttggg acaaaggaag tccctgcgcc ctctcgcacg
    11461 attaccataa aaggcaatgg ctgcggctcg ccgcgcctcg acagccgccg gcgctccggg
    11521 ggccgccgcg ccccyccccc gagcccyccc cggcccgmgg cggccccgcc ccgcccggca
    11581 cccccmcctg ccgccacccc ccgcccggca cggcgagccc cgcgccacgc cccgtacgga
    11641 gccccgcacc cgaagccggg ccgtgctcag caactcgggg aggggggtgc agggggggtt
    11701 gcagcccgac cgacgcgccc acaccccctg ctcacccccc cacgcacaca ccccgcacgc
    11761 agcctttgtt cccctcgcag ccccccccgc accgcggggc accgcccccg gccgcgctcc
    11821 cctcgcgcac actgcggagc gcacaaagcc ccgcgccgcg cccgcagcgc tcacagccgc
    11881 cgggcagcgc ggagccgcac gcggcgctcc ccacgcacac acacacgcac gcaccccccg
    11941 agccgctccc cccgcacaaa gggccctccc ggagcccctc aaggctttca cgcagccaca
    12001 gaaaagaaac aagccgtcat taaaccaagc gctaattaca gcccggagga gaagggccgt
    12061 cccgcccgct cacctgtggg agtaacgcgg tcagtcagag ccggggcggg cggcgcgagg
    12121 cggcggcgga gcggggcacg gggcgaaggc agcgcgcagc gactcccgcc cgccgcgcgc
    12181 ttcgcttttt atagggccgc cgccgccgcc gcctcgccat aaaaggaaac tttcggagcg
    12241 cgccgctctg attggctgcc gccgcmccyc yccsccycsc cccsccccsc cccccscccc
    12301 cccccccccc ccccggcgcg cgcccccccc ccccccccgc ccccatcgct gcacaaaata
    12361 attaaaaaat aaataaatac aaaattgggg gtggggaggg gggggagatg gggagagtga
    12421 agcagaacgt ggggctcacc tcgaccatgg taatagcgat gactaatacg tagatgtact
    12481 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
    12541 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
    12601 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta
    12661 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
    12721 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
    12781 accccgtaat tgattactat taataactag tcaataatca atgtcgagac ccatagagcc
    12841 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
    12901 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
    12961 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
    13021 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac
    13081 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
    13141 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
    13201 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
    13261 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
    13321 gacgagatca tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
    13381 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
    13441 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
    13501 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
    13561 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
    13621 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
    13681 cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
    13741 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
    13801 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
    13861 atcttgttca atggccgatc ccatggtggc ggtaccctcg agcctagggc cgggattctc
    13921 ctccacgtca ccgcatgtta gaagacttcc tctgccctct ccgctgccag atctctcgag
    13981 gccctgtggg aggaagagaa gaggtcagaa gcttataact tcgtggcgcg ccataatgta
    14041 tgctatacga agttatt
//
