LOCUS       sequence-146188-       11467 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses WT SpCas9, EGFP and Puromycin resistance from a CAG 
            promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11467)
  AUTHORS   Saarimaki-Vire J, Balboa D, Russell MA, Saarikettu J, Kinnunen M, 
            Keskitalo S, Malhi A, Valensisi C, Andrus C, Eurola S, Grym H, 
            Ustinov J, Wartiovaara K, Hawkins RD, Silvennoinen O, Varjosalo M, 
            Morgan NG, Otonkoski T
  TITLE     An Activating STAT3 Mutation Causes Neonatal Diabetes through 
            Premature Induction of Pancreatic Differentiation.
  JOURNAL   Cell Rep. 2017 Apr 11;19(2):281-294. doi: 
            10.1016/j.celrep.2017.03.055.
  PUBMED    28402852
REFERENCE   2  (bases 1 to 11467)
  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"; doi: 
            "10.1016/j.celrep.2017.03"; journalName: "Cell Rep"; date: 
            "2017-04-11- 11"; volume: "19"; issue: "2"; pages: "281-294"
COMMENT     Sequence Label: CAG-Cas9-T2A-EGFP-ires-puro
FEATURES             Location/Qualifiers
     source          1..11467
                     /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"
     regulatory      2464..2473
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2473..2538
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2545..2565
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2590..6690
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2590..6690
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6694..6714
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6739..6792
                     /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(6799..6801,6802..6804,6805..7518)
                     /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(6799..6801,6802..6804,6805..7515)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7105..7124)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7452..7473
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(7540..8114,8115..8117,8118..8126)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7707..7724)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7934..7953
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             8127..8726
                     /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(8127..8146)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8623..8643
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(8807..8824)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8813..9037
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(9084..9100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9084..9100)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9097..9119)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9108..9124
                     /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(9132..9138,9139..9156,9157..9162))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9177..9198
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9257..9452
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      9303..9438
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    9458..9592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9660..9677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9831..10419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9911..9930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10590..11381,11382..11450))
                     /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     11213..11232
                     /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 ttcctcgacc
     2461 ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga tattgattac
     2521 aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc
     2581 ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     2641 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     2701 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     2761 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     2821 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     2881 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     2941 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     3001 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     3061 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     3121 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     3181 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     3241 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     3301 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     3361 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     3421 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     3481 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     3541 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     3601 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     3661 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     3721 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     3781 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     3841 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     3901 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     3961 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     4021 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     4081 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     4141 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     4201 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     4261 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     4321 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     4381 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     4441 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     4501 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     4561 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     4621 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     4681 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     4741 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     4801 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     4861 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     4921 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     4981 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     5041 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     5101 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     5161 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     5221 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     5281 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     5341 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     5401 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     5461 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     5521 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     5581 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     5641 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     5701 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     5761 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     5821 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     5881 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     5941 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     6001 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     6061 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     6121 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     6181 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     6241 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     6301 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     6361 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     6421 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     6481 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     6541 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     6601 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     6661 cggatcgacc tgtctcagct gggaggcgac agccccaaga agaagagaaa ggtggaggcc
     6721 agcctcgacg gctccggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     6781 aatcctggcc caatcgatat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     6841 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     6901 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     6961 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     7021 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     7081 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     7141 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     7201 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     7261 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     7321 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     7381 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     7441 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     7501 gacgagctgt acaagtaatt taaagaattc cgcccccccc ccctctccct cccccccccc
     7561 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     7621 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     7681 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     7741 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     7801 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     7861 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     7921 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     7981 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     8041 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     8101 aacacgatga taatatggcc acaaccatga ccgagtacaa gcccacggtg cgcctcgcca
     8161 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     8221 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     8281 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     8341 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     8401 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     8461 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     8521 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     8581 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     8641 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     8701 gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa
     8761 ggagcgcacg accccatgca tcgatgatct agagctcgct gatcagcctc gactgtgcct
     8821 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     8881 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     8941 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     9001 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaacctgc
     9061 agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     9121 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     9181 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     9241 tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     9301 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     9361 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     9421 gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa
     9481 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     9541 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat
     9601 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     9661 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     9721 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     9781 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     9841 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     9901 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     9961 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
    10021 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
    10081 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
    10141 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
    10201 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
    10261 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
    10321 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
    10381 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
    10441 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
    10501 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
    10561 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
    10621 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
    10681 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
    10741 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
    10801 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
    10861 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
    10921 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
    10981 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
    11041 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
    11101 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
    11161 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
    11221 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
    11281 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
    11341 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
    11401 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
    11461 tttcaat
//
