LOCUS       sequence-454866-        9787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1 attB_wildtype site and SpCas9-hygro for integration into Bxb1 
            attP_wildtype landing pad (eg. pHPHS232). Includes pEF1 and attP_GA 
            for second round of recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9787)
  AUTHORS   Nugent PJ, Park H, Wladyka CL, Yelland JN, Sinha S, Chen KY, Bynum 
            C, Quarterman G, Lee SC, Hsieh AC, Subramaniam AR
  TITLE     Decoding post-transcriptional regulatory networks by RNA-linked 
            CRISPR screening in human cells.
  JOURNAL   Nat Methods. 2025 Jun;22(6):1237-1246. doi: 
            10.1038/s41592-025-02702-6. Epub 2025 May 29.
  PUBMED    40442371
REFERENCE   2  (bases 1 to 9787)
  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: 
            "10.1038/s41592-025-02702-6"; journalName: "Nat Methods"; date: 
            "2025-06"; volume: "22"; issue: "6"; pages: "1237-1246"
COMMENT     Sequence Label: pHPHS800
FEATURES             Location/Qualifiers
     source          1..9787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1242..1453
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1454..2392
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2349..2369
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    2408..2455
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    2456..2493
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      2498..2507
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2504..6607
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2504..6607
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6608..6655
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6656..6679
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6695..6748
                     /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             6752..7774
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             6752..7774
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     primer_bind     complement(7822..7841)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7876..7895
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(8032..8823,8824..8892))
                     /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     8655..8674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8893..8997)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      9100..9688
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9589..9608
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcggccgcga gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc
       61 ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc
      121 ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga
      181 tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg
      241 gggaaaaagc tttaggctga aagagagatt tagaatgaca gaatcataga acggcctggg
      301 ttgcaaagga gcacagtgct catccagatc caaccccctg ctatgtgcag ggtcatcaac
      361 cagcagccca ggctgcccag agccacatcc agcctggcct tgaatgcctg cagggatggg
      421 gcatccacag cctccttggg caacctgttc agtgcgtcac caccctctgg gggaaaaact
      481 gcctcctcat atccaaccca aacctcccct gtctcagtgt aaagccattc ccccttgtcc
      541 tatcaagggg gagtttgctg tgacattgtt ggtctggggt gacacatgtt tgccaattca
      601 gtgcatcacg gagaggcaga tcttggggat aaggaagtgc aggacagcat ggacgtggga
      661 catgcaggtg ttgagggctc tgggacactc tccaagtcac agcgttcaga acagccttaa
      721 ggataagaag ataggataga aggacaaaga gcaagttaaa acccagcatg gagaggagca
      781 caaaaaggcc acagacactg ctggtccctg tgtctgagcc tgcatgtttg atggtgtctg
      841 gatgcaagca gaaggggtgg aagagcttgc ctggagagat acagctgggt cagtaggact
      901 gggacaggca gctggagaat tgccatgtag atgttcatac aatcgtcaaa tcatgaaggc
      961 tggaaaagcc ctccaagatc cccaagacca accccaaccc acccaccgtg cccactggcc
     1021 atgtccctca gtgccacatc cccacagttc ttcatcacct ccagggacgg tgaccccccc
     1081 acctccgtgg gcagctgtgc cactgcagca ccgctctttg gagaaggtaa atcttgctaa
     1141 atccagcccg accctcccct ggcacaacgt aaggccatta tctctcatcc aactccagga
     1201 cggagtcagt gagaatattg atatcaacgg tgcccgtcag tgggcagagc gcacatcgcc
     1261 cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg
     1321 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     1381 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     1441 cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta
     1501 tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga
     1561 gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc
     1621 tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca
     1681 ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc
     1741 tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac
     1801 tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg
     1861 ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc
     1921 tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc
     1981 aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc
     2041 tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac
     2101 acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg
     2161 ggcgccgtcc aggcacctcg attagttctc gcgcttttgg agtacgtcgt ctttaggttg
     2221 gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag
     2281 gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg
     2341 gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg
     2401 agctagcggt ttgtctggtc aaccaccgcg gactcagtgg tgtacggtac aaaccggctt
     2461 gtcgacgacg gcggtctccg tcgtcaggat catccgcgcc accatggaca agaagtacag
     2521 catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa
     2581 ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa
     2641 cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag
     2701 aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt
     2761 cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct
     2821 ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt
     2881 ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac
     2941 cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg
     3001 ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat
     3061 ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt
     3121 ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat
     3181 cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct
     3241 gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct
     3301 gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta
     3361 cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct
     3421 gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga
     3481 cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa
     3541 gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg
     3601 agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac
     3661 cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga
     3721 caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca
     3781 ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt
     3841 ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac
     3901 cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc
     3961 ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa
     4021 ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa
     4081 agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa
     4141 ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga
     4201 ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg
     4261 gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt
     4321 cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt
     4381 tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa
     4441 agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct
     4501 gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga
     4561 cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga
     4621 ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa
     4681 tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga
     4741 gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga
     4801 gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga
     4861 gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct
     4921 gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca
     4981 ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt
     5041 tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa
     5101 gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct
     5161 gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg
     5221 cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca
     5281 gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa
     5341 tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt
     5401 ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga
     5461 cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag
     5521 cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga
     5581 gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt
     5641 caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa
     5701 cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt
     5761 gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag
     5821 caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg
     5881 ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt
     5941 ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tactggggat
     6001 caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg
     6061 ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct
     6121 ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact
     6181 ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa
     6241 gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct
     6301 ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa
     6361 tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc
     6421 cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta
     6481 ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac
     6541 cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg
     6601 aggcgacaag cgacctgccg ccacaaagaa ggctggacag gctaagaaga agaaagatta
     6661 caaagacgat gacgataagg gatccggcag cggtgagggc agaggaagtc ttctaacatg
     6721 cggtgacgtg gaggagaatc cgggccctgg taaaaagcct gaactcaccg cgacgtctgt
     6781 cgagaagttt ctgatcgaaa agttcgacag cgtgtccgac ctgatgcagc tctcggaggg
     6841 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     6901 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     6961 gctcccgatt ccggaagtgc ttgacattgg ggaatttagc gagagcctga cctattgcat
     7021 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     7081 tctgcaaccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     7141 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     7201 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     7261 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     7321 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     7381 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     7441 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     7501 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     7561 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     7621 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     7681 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     7741 acgccccagc actcgtccga gggcaaagga atgatcagag agatcctcgc ggccgcggat
     7801 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     7861 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     7921 aataaactct agagagtgat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     7981 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     8041 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     8101 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     8161 aatgataccg cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc
     8221 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     8281 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     8341 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     8401 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     8461 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     8521 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     8581 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     8641 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     8701 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     8761 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     8821 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     8881 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     8941 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     9001 atttccccga aaagtgccac ctgtcatgac caaaatccct taacgtgagt tttcgttcca
     9061 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     9121 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     9181 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     9241 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     9301 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     9361 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     9421 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     9481 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     9541 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     9601 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     9661 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     9721 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     9781 taaccgt
//
