LOCUS       sequence-444492-       14518 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Targeting vector for CRISPRd generation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14518)
  AUTHORS   Matsui S, Granitto M, Buckley M, Ludwig K, Koigi S, Shiley J, 
            Zacharias WJ, Mayhew CN, Lim HW, Iwafuchi M
  TITLE     Pioneer and PRDM transcription factors coordinate bivalent 
            epigenetic states to safeguard cell fate.
  JOURNAL   Mol Cell. 2024 Jan 4:S1097-2765(23)01026-2. doi: 
            10.1016/j.molcel.2023.12.007.
  PUBMED    38211589
REFERENCE   2  (bases 1 to 14518)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2024 Jan 4:S1097-2765(23)01026-2. doi: 
            10.1016/j.molcel.2023.12.007."
COMMENT     Sequence Label: pAAVS1-NDi-CRISPRd (ABI-dCas9-mCherry)
FEATURES             Location/Qualifiers
     source          1..14518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(54..122,123..914)
                     /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(272..291)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1085..1673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1574..1593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    1721..2557
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    2590..2608
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2626..2644
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2662..2680
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2698..2716
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2734..2752
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2770..2788
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2806..2824
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2869..2893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(3897..3923,3924..3926,3927..6413,6414..6416,
                     6417..8000)
                     /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             3897..8000
                     /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             8004..8030
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             8010..8036
                     /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             8049..8069
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8076..8096
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8145..8201
                     /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             8202..8912
                     /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(8347..8365)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     8615..8634
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    8944..9065
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8981..9000)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9035..9054
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9114..9130)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9114..9130)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9127..9149)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9138..9154
                     /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(9162..9168,9169..9186,9187..9192))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9207..9228
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9345..9362)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9418..9451
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     9782..9801
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    9800..9855
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     9901..9920
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(10011..10757)
                     /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(10789..10808)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          10816..11832
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(10870..10892)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(11833..12109)
                     /label=chicken beta-actin promoter
     enhancer        12111..12490
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    12496..12720
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     12709..12726
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(12746..13549)
                     /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(12857..12876)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     13467..13486
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     regulatory      13546..13555
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(13571..13624)
                     /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    13648..13673
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    13680..13713
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    13715..14518
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 aagggcgaat tcaaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
       61 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
      361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
      421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
      481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
      541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
      601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
      661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
      721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
      781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
      841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
      901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
      961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1021 tcccttaacg ggagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1201 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     1501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     1561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     1621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     1681 gcaacgcggc ctttttacgg ttcctggcga attcgccctt agagcagagc caggaacccc
     1741 tgtagggaag gggcaggaga gccaggggca tgagatggtg gacgaggaag ggggacaggg
     1801 aagcctgagc gcctctcctg ggcttgccaa ggactcaaac ccagaagccc agagcagggc
     1861 cttagggaag cgggaccctg ctctgggcgg aggaatatgt cccagatagc actggggact
     1921 ctttaaggaa agaaggatgg agaaagagaa agggagtaga ggcggccacg acctggtgaa
     1981 cacctaggac gcaccattct cacaaaggga gttttccaca cggacacccc cctcctcacc
     2041 acagccctgc caggacgggg ctggctactg gccttatctc acaggtaaaa ctgacgcacg
     2101 gaggaacaat ataaattggg gactagaaag gtgaagagcc aaagttagaa ctcaggacca
     2161 acttattctg attttgtttt tccaaactgc ttctcctctt gggaagtgta aggaagctgc
     2221 agcaccagga tcagtgaaac gcaccagaca gccgcgtcag agcagctcag gttctgggag
     2281 agggtagcgc agggtggcca ctgagaaccg ggcaggtcac gcatcccccc cttccctccc
     2341 accccctgcc aagctctccc tcccaggatc ctctctggct ccatcgtaag caaaccttag
     2401 aggttctggc aaggagagag atggctccag gaaatggggg tgtgtcacca gataaggaat
     2461 ctgcctaaca ggaggtgggg gttagaccca atatcaggag actaggaagg aggaggccta
     2521 aggatggggc ttttctgtca ccaatcctgt ccctagtaaa gctgatatct ccaccccaca
     2581 gtggtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     2641 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     2701 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     2761 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
     2821 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
     2881 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
     2941 ccacttccta ccctcgtaaa cttaaggcca ccatgacgcg tgtgcctttg tatggtttta
     3001 cttcgatttg tggaagaaga cctgagatgg aagctgctgt ttcgactata ccaagattcc
     3061 ttcaatcttc ctctggttcg atgttagatg gtcggtttga tcctcaatcc gccgctcatt
     3121 tcttcggtgt ttacgacggc catggcggtt ctcaggtagc gaactattgt agagagagga
     3181 tgcatttggc tttggcggag gagatagcta aggagaaacc gatgctctgc gatggtgata
     3241 cgtggctgga gaagtggaag aaagctcttt tcaactcgtt cctgagagtt gactcggaga
     3301 ttgagtcagt tgcgccggag acggttgggt caacgtcggt ggttgccgtt gttttcccgt
     3361 ctcacatctt cgtcgctaac tgcggtgact ctagagccgt tctttgccgc ggcaaaactg
     3421 cacttccatt atccgttgac cataaaccgg atagagaaga tgaagctgcg aggattgaag
     3481 ccgcaggagg gaaagtgatt cagtggaatg gagctcgtgt tttcggtgtt ctcgccatgt
     3541 cgagatccat tggcgataga tacttgaaac catccatcat tcctgatccg gaagtgacgg
     3601 ctgtgaagag agtaaaagaa gatgattgtc tgattttggc gagtgacggg gtttgggatg
     3661 taatgacgga tgaagaagcg tgtgagatgg caaggaagcg gattctcttg tggcacaaga
     3721 aaaacgcggt ggctggggat gcatcgttgc tcgcggatga gcggagaaag gaagggaaag
     3781 atcctgcggc gatgtccgcg gctgagtatt tgtcaaagct ggcgatacag agaggaagca
     3841 aagacaacat aagtgtggtg gtggttgatt tgaagcatat gggtggaggc tccgggatgg
     3901 acaagaagta cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca
     3961 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     4021 gcatcaagaa gaacctgatc ggcgccctgc tgttcgacag cggagaaaca gccgaggcca
     4081 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     4141 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     4201 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     4261 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     4321 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     4381 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     4441 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     4501 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     4561 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga
     4621 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     4681 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     4741 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     4801 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     4861 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     4921 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     4981 acatcgatgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     5041 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     5101 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     5161 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     5221 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     5281 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     5341 tggacaaggg cgccagcgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     5401 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtaca
     5461 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     5521 gcgagcagaa aaaagccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
     5581 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
     5641 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
     5701 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
     5761 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
     5821 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
     5881 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
     5941 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
     6001 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
     6061 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
     6121 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
     6181 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
     6241 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
     6301 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
     6361 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gacgctatcg
     6421 tgcctcagag ctttctgaag gacgactcca tcgataacaa agtgctgact cggagcgaca
     6481 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
     6541 actggcgcca gctgctgaat gccaagctga ttacccagag gaagttcgac aatctgacca
     6601 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
     6661 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
     6721 agtacgacga gaacgacaaa ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
     6781 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
     6841 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
     6901 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
     6961 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     7021 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
     7081 tgatcgagac aaacggcgaa acaggcgaga tcgtgtggga taagggccgg gactttgcca
     7141 ccgtgcggaa agtgctgtct atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
     7201 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgacaag ctgatcgcca
     7261 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
     7321 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
     7381 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
     7441 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
     7501 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
     7561 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
     7621 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
     7681 acaaacacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
     7741 tggccgacgc taatctggac aaggtgctga gcgcctacaa caagcacaga gacaagccta
     7801 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
     7861 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
     7921 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
     7981 tgtctcagct gggaggcgac gcctatccct atgacgtgcc cgattatgcc agcctgggca
     8041 gcggctcccc caagaaaaaa cgcaaggtgg aagatcctaa gaaaaagcgg aaagtggacg
     8101 gcattggtag tgggagcaac ggcagcagcg gatccggaag cggagctact aacttcagcc
     8161 tgctgaagca ggctggagac gtggaggaga accctggacc tatggtgagc aagggcgagg
     8221 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     8281 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     8341 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     8401 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     8461 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     8521 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     8581 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     8641 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     8701 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     8761 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     8821 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     8881 ccaccggcgg catggacgag ctgtacaagt aaaccggtgc ggccgcaatc gatcgcccac
     8941 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     9001 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     9061 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     9121 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     9181 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     9241 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     9301 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     9361 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaagga cggccgcata
     9421 acttcgtata atgtatgcta tacgaagtta taagcttggg ctgcaggtcg agggatctcc
     9481 ataagagaag agggacagct atgactggga gtagtcagga gaggaggaaa aatctggcta
     9541 gtaaaacatg taaggaaaat tttagggatg ttaaagaaaa aaataacaca aaacaaaata
     9601 taaaaaaaat ctaacctcaa gtcaaggctt ttctatggaa taaggaatgg acagcagggg
     9661 gctgtttcat atactgatga cctctttata gccaaccttt gttcatggca gccagcatat
     9721 gggcatatgt tgccaaactc taaaccaaat actcattctg atgttttaaa tgatttgccc
     9781 tcccatatgt ccttccgagt gagagacaca aaaaattcca acacactatt gcaatgaaaa
     9841 taaatttcct ttattagcca gaagtcagat gctcaagggg cttcatgatg tccccataat
     9901 ttttggcaga gggaaaaaga tctcagtggt atttgtgagc cagggcattg gccacaccag
     9961 ccaccacctt ctgataggca gcctgcacct gaggagtgaa ttcacgcgtt ttagcctggc
    10021 agcatatcga gatcgaagtc atcgagagcg tcagcaggga gcatatccag atcaaaatca
    10081 tccagggcat cggcgggcag catgtccagg tcgaagtcgt ccagggcgtc ggtgggtccg
    10141 ccgctctcgc acttcagctg cttctcgagg ccgcagatga tcagttccag gccgaacagg
    10201 aaggcgggct cggcgccctg tctgtcgaac agctcgatgg cctgcttcag cagggggggc
    10261 atgctgtcgg tggtgggtgt ctctctttcc tctttggcga cctggtgttc ctgttcttcc
    10321 agcacgcagc ccagggtgaa gtggcccacg gcgctcaggg cgtacagggc gttttccagg
    10381 ctgaagccct gctggcacag gaaggccagc tggttttcca gtgtctcgta ctgcttctcg
    10441 gtgggtctgg tgcccaggtg cactttggcg ccgtcccggt ggctcagcag ggcgcatctg
    10501 tagctcttgg cgttgttccg caggaagtcc tgccagctct cgccttccag agggcagctg
    10561 tgggtgtggt gccggtccag catctcgatg ggcagggcgt ccagcagggc ccgcttgttc
    10621 ttcacgtgcc agtacagggt gggctgttcc acgcccagtt tctgggccag cttccgggtg
    10681 gtcaggccct cgatgcccac gccgttcagc agttccaggg cgctgttgat cactttgctc
    10741 ttgtccagcc ggctcatgtc gacattcttt gccaaaatga tgagacagca caataaccag
    10801 cacgttgccc aggagctgta ggaaaaagaa gaaggcatga acatggttag cagaggctct
    10861 agagccgccg gtcacacgcc agaagccgaa ccccgccctg ccccgtcccc cccgaaggca
    10921 gccgtccccc cgcggacagc cccgaggctg gagagggaga aggggacggc ggcgcggcga
    10981 cgcacgaagg ccctccccgc ccatttcctt cctgccggcg ccgcaccgct tcgccccgcg
    11041 cccgctagag ggggtgcggc ggcgcctccc agatttcggc tccgcacaga tttgggacaa
    11101 aggaagtccc tgcgccctct cgcacgatta ccataaaagg caatggctgc ggctcgccgc
    11161 gcctcgacag ccgccggcgc tccgggggcc gccgcgcccc tcccccgagc cctccccggc
    11221 ccgaggcggc cccgccccgc ccggcacccc cacctgccgc caccccccgc ccggcacggc
    11281 gagccccgcg ccacgccccg tacggagccc cgcacccgaa gccgggccgt gctcagcaac
    11341 tcggggaggg gggtgcaggg ggggttgcag cccgaccgac gcgcccacac cccctgctca
    11401 cccccccacg cacacacccc gcacgcagcc tttgttcccc tcgcagcccc ccccgcaccg
    11461 cggggcaccg cccccggccg cgctcccctc gcgcacactg cggagcgcac aaagccccgc
    11521 gccgcgcccg cagcgctcac agccgccggg cagcgcggag ccgcacgcgg cgctccccac
    11581 gcacacacac acgcacgcac cccccgagcc gctccccccg cacaaagggc cctcccggag
    11641 cccctcaagg ctttcacgca gccacagaaa agaaacaagc cgtcattaaa ccaagcgcta
    11701 attacagccc ggaggagaag ggccgtcccg cccgctcacc tgtgggagta acgcggtcag
    11761 tcagagccgg ggcgggcggc gcgaggcggc ggcggagcgg ggcacggggc gaaggcagcg
    11821 cgcagcgact cccgcccgcc gcgcgcttcg ctttttatag ggccgccgcc gccgccgcct
    11881 cgccataaaa ggaaactttc ggagcgcgcc gctctgattg gctgccgccg cacctctccg
    11941 cctcgccccg ccccgcccct cgccccgccc cgccccgcct ggcgcgcgcc cccccccccc
    12001 cccgccccca tcgctgcaca aaataattaa aaaataaata aatacaaaat tgggggtggg
    12061 gagggggggg agatggggag agtgaagcag aacgtggggc tcacctcgac catggtaata
    12121 gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg
    12181 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata
    12241 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt
    12301 caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg
    12361 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac
    12421 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat
    12481 aatcaatgtc gagacccata gagcccaccg catccccagc atgcctgcta ttgtcttccc
    12541 aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca
    12601 gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca
    12661 gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag
    12721 tcgaggctga tcagcgggtt taaactcaga agaactcgtc aagaaggcga tagaaggcga
    12781 tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc
    12841 cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca
    12901 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg
    12961 gcaagcaggc atcgccatgg gtcacgacga gatcatcgcc gtcgggcatg cgcgccttga
    13021 gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat
    13081 cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt
    13141 cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg
    13201 atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca
    13261 atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc
    13321 ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg
    13381 acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg
    13441 catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag
    13501 cggccggaga acctgcgtgc aatccatctt gttcaatggc cgatcccatg gtggcggtac
    13561 cctcgagcct agggccggga ttctcctcca cgtcaccgca tgttagaaga cttcctctgc
    13621 cctctccgct gccagatctc tcgaggccct gtgggaggaa gagaagaggt cagaagctta
    13681 taacttcgta taatgtatgc tatacgaagt tattgcccca ctgtggggtg gaggggacag
    13741 ataaaagtac ccagaaccag agccacatta accggccctg ggaatataag gtggtcccag
    13801 ctcggggaca caggatccct ggaggcagca aacatgctgt cctgaagtgg acataggggc
    13861 ccgggttgga ggaagaagac tagctgagct ctcggacccc tggaagatgc catgacaggg
    13921 ggctggaaga gctagcacag actagagagg taaggggggt aggggagctg cccaaatgaa
    13981 aggagtgaga ggtgacccga atccacagga gaacggggtg tccaggcaaa gaaagcaaga
    14041 ggatggagag gtggctaaag ccagggagac ggggtacttt ggggttgtcc agaaaaacgg
    14101 tgatgatgca ggcctacaag aaggggaggc gggacgcaag ggagacatcc gtcggagaag
    14161 gccatcctaa gaaacgagag atggcacagg ccccagaagg agaaggaaaa gggaacccag
    14221 cgagtgaaga cggcatgggg ttgggtgagg gaggagagat gcccggagag gacccagaca
    14281 cggggaggat ccgctcagag gacatcacgt ggtgcagcgc cgagaaggaa gtgctccgga
    14341 aagagcatcc ttgggcagca acacagcaga gagcaagggg aagagggagt ggaggaagac
    14401 ggaacctgaa ggaggcggca gggaaggatc tgggccagcc gtagaggtga cccaggccac
    14461 aagctgcaga cagaaagcgg cacaggccca ggggagagaa tgctggtcag agaaagca
//
