LOCUS       sequence-314866-        8965 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Cas9-2A-Blast and a sgRNA excising EGFP flanked by a 
            splice acceptor and a splice donor from the 
            Intron-Tagging-EGFP-Donor plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8965)
  AUTHORS   Reicher A, Koren A, Kubicek S
  TITLE     Pooled protein tagging, cellular imaging, and in situ sequencing for
            monitoring drug action in real time.
  JOURNAL   Genome Res. 2020 Dec;30(12):1846-1855. doi: 10.1101/gr.261503.120. 
            Epub 2020 Nov 17.
  PUBMED    33203764
REFERENCE   2  (bases 1 to 8965)
  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: 
            "10.1101/gr.261503.120"; journalName: "Genome Res"; date: "2020-12";
            volume: "30"; issue: "12"; pages: "1846-1855"
COMMENT     Sequence Label: Intron-Tagging-pX330-Cas9-Blast
FEATURES             Location/Qualifiers
     source          1..8965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..37)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    26..233
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   242..382
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   242..371
                     /label=AAV2 ITR
     rep_origin      457..912
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(544..563)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     754..775
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(929..948)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1048..1070
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1108..1126)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1194..1298
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1299..1367,1368..2159)
                     /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(1517..1536)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2330..2918
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2819..2838
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2980..3228
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2980..3000
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3151..3170
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3249..3324
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        3421..3706
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        3708..3985
                     /label=chicken beta-actin promoter
     intron          3986..4213
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      4225..4234
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4234..4299
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4306..4326
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4351..8451
                     /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             4351..8451
                     /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             8452..8499
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8509..8565
                     /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             8566..8964
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
ORIGIN
        1 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
       61 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
      121 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
      181 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
      241 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
      301 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
      361 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
      421 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
      481 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
      541 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      601 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      661 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      721 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      781 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
      841 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      901 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
      961 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     1021 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     1081 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     1141 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     1201 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1261 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1321 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1381 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1441 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1501 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1561 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1621 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1681 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1741 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1801 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     1861 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1921 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1981 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     2041 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2101 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2161 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2221 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2281 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2341 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2401 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2461 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2521 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2581 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2641 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2701 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2761 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2821 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2881 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2941 tacggttcct ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt
     3001 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     3061 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     3121 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     3181 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga gatcgagtgc
     3241 cgcatcacgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     3301 gaaaaagtgg caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat
     3361 aaggctagtc cgtttttagc gcgtgcgcca attctgcaga caaatggctc tagaggtacc
     3421 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     3481 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3541 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3601 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt
     3661 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
     3721 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
     3781 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccggg
     3841 gggggggggg sggggsgrgg ggsggggsgg ggsgaggcgg agaggtgcgg cggcagccaa
     3901 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
     3961 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
     4021 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
     4081 gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt
     4141 taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat
     4201 cacttttttt caggttggac cggtgccacc atggactata aggaccacga cggagactac
     4261 aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
     4321 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
     4381 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     4441 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     4501 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     4561 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     4621 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     4681 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     4741 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     4801 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     4861 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     4921 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     4981 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     5041 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     5101 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     5161 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     5221 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     5281 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     5341 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     5401 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     5461 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     5521 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     5581 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     5641 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     5701 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     5761 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     5821 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     5881 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     5941 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     6001 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     6061 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     6121 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     6181 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     6241 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     6301 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     6361 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     6421 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     6481 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     6541 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     6601 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     6661 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     6721 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     6781 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     6841 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     6901 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     6961 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     7021 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     7081 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     7141 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     7201 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     7261 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     7321 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     7381 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     7441 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     7501 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     7561 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     7621 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     7681 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     7741 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     7801 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     7861 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     7921 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     7981 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     8041 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     8101 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     8161 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     8221 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     8281 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     8341 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     8401 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg
     8461 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gatccggcgc aacaaacttc
     8521 tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgatggc caagcctttg
     8581 tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc
     8641 tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt
     8701 gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct
     8761 gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc
     8821 atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa
     8881 gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc
     8941 tctggttatg tgtgggaggg ctaag
//
