LOCUS       sequence-256469-       12420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EF1a promoter expresses SpyoCas9 and P2A provides EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12420)
  AUTHORS   DeWeirdt PC, Sangree AK, Hanna RE, Sanson KR, Hegde M, Strand C, 
            Persky NS, Doench JG
  TITLE     Genetic screens in isogenic mammalian cell lines without single cell
            cloning.
  JOURNAL   Nat Commun. 2020 Feb 6;11(1):752. doi: 10.1038/s41467-020-14620-6.
  PUBMED    32029722
REFERENCE   2  (bases 1 to 12420)
  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"; journalName: "Nat 
            Commun."; date: "2020-02-6"; volume: "11(1):752. doi"; pages: " 
            10.1038/s41467-020-14620-6"
COMMENT     Sequence Label: pXPR_124
FEATURES             Location/Qualifiers
     source          1..12420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..229
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             230..410
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    457..582
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1075..1308
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1493..1537
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1803..1920
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    1938..1958
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        1959..3137
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2190..3128
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3085..3105
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    3137..3161
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      3162..3171
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3171..3236
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3243..3263
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3288..7388
                     /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             3288..7388
                     /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             7389..7436
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7446..7502
                     /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             join(7503..7505,7506..7508,7509..8222)
                     /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(7503..7505,7506..7508,7509..8219)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7809..7828)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8156..8177
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(8223..8247)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    8265..8853
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8318..8338)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8736..8747)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8941..9174
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9246..9367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9283..9302)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9337..9356
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9407..9542
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9562..9581)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9563..9581)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9591..9608)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9591..9607)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9600..9622)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9749..10204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9836..9855)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10046..10067
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10230..10334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10335..10403,10404..11195)
                     /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(10553..10572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11366..11954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11855..11874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12108..12125
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12242..12263
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12278..12283,12284..12301,12302..12308)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12316..12332
                     /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)."
     primer_bind     12321..12343
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12340..12356
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12340..12356
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     12375..12395
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        12377..12395
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
       61 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      121 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      181 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      241 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      301 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      361 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      421 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      481 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      541 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      601 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      661 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
      721 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
      781 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
      841 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
      901 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
      961 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     1021 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     1081 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     1141 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     1201 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     1261 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     1321 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     1381 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta
     1801 gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     1861 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     1921 actagtgatt atcggatcaa ctttgtatag aaaagttggg ctccggtgcc cgtcagtggg
     1981 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
     2041 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     2101 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     2161 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
     2221 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
     2281 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
     2341 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     2401 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     2461 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     2521 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     2581 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     2641 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg tctcgcgccg ccgtgtatcg
     2701 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     2761 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     2821 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     2881 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     2941 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     3001 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     3061 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     3121 catttcaggt gtcgtgacaa gtttgtacaa aaaagcaggc tgccaccatg gactataagg
     3181 accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg
     3241 ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca
     3301 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     3361 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     3421 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
     3481 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     3541 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     3601 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     3661 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     3721 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     3781 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     3841 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     3901 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     3961 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc
     4021 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     4081 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     4141 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     4201 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     4261 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     4321 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     4381 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     4441 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     4501 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     4561 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     4621 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     4681 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     4741 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     4801 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     4861 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     4921 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     4981 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     5041 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     5101 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     5161 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     5221 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     5281 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     5341 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     5401 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     5461 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     5521 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     5581 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     5641 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     5701 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     5761 aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct
     5821 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
     5881 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
     5941 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
     6001 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     6061 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     6121 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     6181 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     6241 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     6301 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     6361 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     6421 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     6481 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
     6541 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     6601 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
     6661 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     6721 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     6781 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     6841 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     6901 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
     6961 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
     7021 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
     7081 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
     7141 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
     7201 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
     7261 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
     7321 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
     7381 gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggaa
     7441 gcggagccac gaacttctct ctgttaaagc aagcaggaga tgttgaagaa aaccccgggc
     7501 ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     7561 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     7621 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     7681 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     7741 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     7801 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     7861 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     7921 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     7981 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     8041 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     8101 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     8161 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     8221 aaacccagct ttcttgtaca aagtggtgat aatcgaattc cgataatcaa cctctggatt
     8281 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     8341 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     8401 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     8461 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     8521 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     8581 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     8641 ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct
     8701 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     8761 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     8821 cgagtcggat ctccctttgg gccgcctccc cgcatcggga attcccgcgg ttcgctttaa
     8881 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     8941 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     9001 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     9061 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     9121 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta
     9181 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     9241 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     9301 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     9361 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     9421 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     9481 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     9541 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     9601 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     9661 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     9721 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     9781 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     9841 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     9901 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     9961 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
    10021 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
    10081 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
    10141 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
    10201 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
    10261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
    10321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
    10381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
    10441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
    10501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
    10561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
    10621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
    10681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
    10741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
    10801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
    10861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
    10921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
    10981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
    11041 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
    11101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
    11161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
    11221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
    11281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
    11341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
    11401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
    11461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
    11521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
    11581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
    11641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
    11701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
    11761 gagaaagcgc cacgcttccc gaagagagaa aggcggacag gtatccggta agcggcaggg
    11821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
    11881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
    11941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
    12001 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
    12061 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
    12121 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
    12181 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
    12241 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
    12301 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
    12361 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt
//
