LOCUS       sequence-308993-        8972 bp    DNA     circular SYN 15-DEC-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 8972)
  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 8972)
  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: 
            "10.1101/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..8972
                     /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        2986..3234
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2986..3006
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3157..3176
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3255..3330
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        3427..3712
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        3714..3991
                     /label=chicken beta-actin promoter
     intron          3992..4220
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      4232..4241
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4241..4306
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4313..4333
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4358..8458
                     /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             4358..8458
                     /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             8459..8506
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8516..8572
                     /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             8573..8971
                     /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 gccctagcgc
      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 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
      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 tactgtcctt 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 ctcacatgtc cccacgaggg cctatttccc
     3001 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     3061 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     3121 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     3181 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgagatc
     3241 gagtgccgca tcacgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     3301 caacttgaaa aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt
     3361 taaaataagg ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa tggctctaga
     3421 ggtacccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     3481 cccattgacg tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     3541 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     3601 tattgacgtc aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg
     3661 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt
     3721 gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta
     3781 tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc
     3841 gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc
     3901 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
     3961 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgacgctgc cttcgccccg
     4021 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
     4081 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
     4141 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
     4201 ctgaaatcac tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg
     4261 agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa
     4321 gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct
     4381 ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
     4441 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
     4501 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag
     4561 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga
     4621 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga
     4681 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca
     4741 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
     4801 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct
     4861 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt
     4921 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa
     4981 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct
     5041 gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac
     5101 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
     5161 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
     5221 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa
     5281 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca
     5341 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga
     5401 gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca
     5461 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
     5521 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag
     5581 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt
     5641 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc
     5701 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag
     5761 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca
     5821 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc
     5881 caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata
     5941 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt
     6001 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt
     6061 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc
     6121 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa
     6181 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag
     6241 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa
     6301 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg
     6361 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc
     6421 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca
     6481 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg
     6541 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
     6601 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac
     6661 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa
     6721 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga
     6781 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga
     6841 catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga
     6901 cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa
     6961 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc
     7021 caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag
     7081 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa
     7141 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct
     7201 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga
     7261 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
     7321 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
     7381 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat
     7441 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga
     7501 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac
     7561 cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat
     7621 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
     7681 tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa
     7741 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt
     7801 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat
     7861 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga
     7921 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg
     7981 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc
     8041 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc
     8101 cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat
     8161 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa
     8221 agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat
     8281 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac
     8341 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
     8401 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa
     8461 aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat ccggcgcaac
     8521 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgatggccaa
     8581 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
     8641 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
     8701 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
     8761 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
     8821 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
     8881 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
     8941 gctgccctct ggttatgtgt gggagggcta ag
//
