LOCUS       sequence-122831-       12561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DHFR destabilised domain (DD) fused to dCas9VP192 (S.pyogenes) on 
            CAG expression vector. DDdCas9VP192 protein is stabilised by 
            Trimethoprim..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12561)
  AUTHORS   Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski 
            T
  TITLE     Conditionally Stabilized dCas9 Activator for Controlling Gene 
            Expression in Human Cell Reprogramming and Differentiation.
  JOURNAL   Stem Cell Reports. 2015 Sep 8;5(3):448-59. doi: 
            10.1016/j.stemcr.2015.08.001.
  PUBMED    26352799
REFERENCE   2  (bases 1 to 12561)
  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"; doi: 
            "10.1016/j.stemcr.2015.08"; journalName: "Stem Cell Reports"; date: 
            "2015-09-8- 8"; volume: "5"; issue: "3"; pages: "448-59"
COMMENT     Sequence Label: CAG-DDdCas9VP192-T2A-EGFP-ires-puro
FEATURES             Location/Qualifiers
     source          1..12561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    576..710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        724..1103
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1105..1382
                     /label=chicken beta-actin promoter
     intron          1383..2399
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2323..2345
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2407..2426
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2466..2942
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             2967..2993
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2997..3017
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3027..3050,3051..3053,3054..5609,5610..5612,
                     5613..7127)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(3027..3050,3051..3053,3054..5540,5541..5543,
                     5544..7127)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7131..7151
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7188..7298
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             7350..7460
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             7512..7622
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             7674..7784
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             7824..7877
                     /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             join(7884..7886,7887..7889,7890..8603)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(7884..7886,7887..7889,7890..8600)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8190..8209)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8537..8558
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(8634..9208,9209..9211,9212..9220)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8801..8818)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     9028..9047
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             9221..9820
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(9221..9240)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9717..9737
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(9901..9918)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9907..10131
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(10178..10194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10178..10194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10191..10213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10202..10218
                     /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(10226..10232,10233..10250,10251..10256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10271..10292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        10351..10546
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      10397..10532
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    10552..10686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10754..10771)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10925..11513)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11005..11024)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11684..12475,12476..12544))
                     /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     12307..12326
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaccgctttg gaaaaacaaa gactgtattt cctggaaatt aatgtttatt caataaactg
       61 tgtattcagc tatattcaca tagtggtgag gctgaaatga ggcgggaaga ggcggttggg
      121 gcttaattat atcaatttgg gtggccccac agcgcctcca aggcgccagt cctgttttga
      181 caagttgcct ctggaagcct ctcttctttt tctccagagt aagcggaggc caggggcccc
      241 cggcctctgc ttaatactaa aaaaaacagc tgttgtcata gtaatgattg ggtggaaaca
      301 ttccaggcct gggtggagag gctttttgct tcctcttgca aaaccacact gacattccag
      361 gcctgggtgg agaggctttt tgcttcctct tgcaaaacca cactgccctc tggagggcag
      421 ttgcctagca actaattaaa agaggatgtc gcacggccag ctgcggtcag ttagtcactt
      481 cctgcttaac tgacttgaca ttttctattt taagagtcgg gaggaaaatt actgtgttgg
      541 aggccctccg ccatcttctg aagctgaatc gaattaactt gtttattgca gcttataatg
      601 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
      661 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgatatcatc
      721 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      781 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      841 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      901 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      961 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1021 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1081 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     1141 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     1201 cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
     1261 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     1321 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     1381 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1441 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1501 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
     1561 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
     1621 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
     1681 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1741 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1801 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1861 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1921 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1981 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     2041 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     2101 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     2161 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     2221 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     2281 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     2341 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcntacagc
     2401 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgggatccg
     2461 ccaccatgat cagtctgatt gcggcgttag cggtagatta cgttatcggc atggaaaacg
     2521 ccatgccgtg gaacctgcct gccgatctcg cctggtttaa acgcaacacc ttaaataaac
     2581 ccgtgattat gggccgccat acctgggaat caatcggtcg tccgttgcca ggacgcaaaa
     2641 atattatcct cagcagtcaa ccgagtacgg acgatcgcgt aacgtgggtg aagtcggtgg
     2701 atgaagccat cgcggcgtgt ggtgacgtac cagaaatcat ggtgattggc ggcggtcgcg
     2761 ttattgaaca gttcttgcca aaagcgcaaa aactgtatct gacgcatatc gacgcagaag
     2821 tggaaggcga cacccatttc ccggattacg agccggatga ctgggaatcg gtattcagcg
     2881 aattccacga tgctgatgcg cagaactctc acagctattg ctttgagatt ctggagcggc
     2941 gaggctccgg aaccggtgcc accatgtacc catacgatgt tccagattac gcttcgccga
     3001 agaaaaagcg caaggtcgaa gcgtccgaca agaagtacag catcggcctg gccatcggca
     3061 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     3121 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     3181 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     3241 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
     3301 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
     3361 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
     3421 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
     3481 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
     3541 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     3601 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
     3661 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
     3721 agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc cccaacttca
     3781 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     3841 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     3901 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     3961 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     4021 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     4081 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     4141 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     4201 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     4261 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     4321 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     4381 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     4441 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     4501 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     4561 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     4621 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
     4681 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
     4741 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
     4801 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
     4861 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     4921 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
     4981 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
     5041 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
     5101 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
     5161 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
     5221 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
     5281 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
     5341 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
     5401 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
     5461 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
     5521 tgtccgacta cgatgtcgac gccatcgtgc ctcagagctt tctgaaggac gactccatcg
     5581 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
     5641 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
     5701 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
     5761 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
     5821 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
     5881 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
     5941 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
     6001 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
     6061 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
     6121 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
     6181 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
     6241 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
     6301 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
     6361 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
     6421 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
     6481 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
     6541 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
     6601 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
     6661 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
     6721 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
     6781 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
     6841 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
     6901 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
     6961 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
     7021 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
     7081 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacagc cccaagaaga
     7141 agagaaaggt ggaggccagc ctcgagtgcg aaagcggcgg gccggccgac gcccttgacg
     7201 attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt gatatgctgc
     7261 ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtcacta cccagcgcta
     7321 gcgtcgagtg cgaaagcggc gggccggccg acgcccttga cgattttgac ttagacatgc
     7381 tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac gctcttgacg
     7441 attttgacct tgacatgctc cccgggtcac tacccagcgc tagcgtcgag tgcgaaagcg
     7501 gcgggccggc cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg
     7561 acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc
     7621 tccccgggtc actacccagc gctagcgtcg agtgcgaaag cggcgggccg gccgacgccc
     7681 ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata
     7741 tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg tcactaccca
     7801 gcgctagcgt cgacggctcc ggagagggca gaggaagtct gctaacatgc ggtgacgtcg
     7861 aggagaatcc tggcccaatc gatatggtga gcaagggcga ggagctgttc accggggtgg
     7921 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     7981 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     8041 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     8101 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     8161 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     8221 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     8281 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     8341 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     8401 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     8461 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     8521 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     8581 gcatggacga gctgtacaag taatttaaag aattcccgcc cccccccccc ccccccctct
     8641 ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt
     8701 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct
     8761 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa
     8821 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg
     8881 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc
     8941 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg
     9001 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg
     9061 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc
     9121 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg
     9181 gttttccttt gaaaaacacg atgataatat ggccacaacc atgaccgagt acaagcccac
     9241 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     9301 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     9361 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     9421 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     9481 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     9541 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     9601 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     9661 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     9721 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     9781 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca
     9841 gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg atctagagct cgctgatcag
     9901 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     9961 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
    10021 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
    10081 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
    10141 cggaaagaac ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
    10201 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
    10261 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
    10321 agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc
    10381 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
    10441 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
    10501 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt
    10561 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
    10621 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
    10681 tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
    10741 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
    10801 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
    10861 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
    10921 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
    10981 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
    11041 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
    11101 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
    11161 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
    11221 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
    11281 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
    11341 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
    11401 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
    11461 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
    11521 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
    11581 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
    11641 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
    11701 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
    11761 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
    11821 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
    11881 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
    11941 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
    12001 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
    12061 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
    12121 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
    12181 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
    12241 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
    12301 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
    12361 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
    12421 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
    12481 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
    12541 tcatactctt cctttttcaa t
//
