LOCUS       sequence-474355-       14959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ef1a-DivA-BE3-T2A-tagBFP-wPRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14959)
  AUTHORS   Schwartz CI, Abell NS, Li A, Aradhana, Tycko J, Truong A, Montgomery
            SB, Hess GT
  TITLE     Toward optimizing diversifying base editors for high-throughput 
            mutational scanning studies.
  JOURNAL   Nucleic Acids Res. 2025 Jun 20;53(12):gkaf620. doi: 
            10.1093/nar/gkaf620.
  PUBMED    40613705
REFERENCE   2  (bases 1 to 14959)
  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: "Nucleic 
            Acids Res."; date: "2025-06-20"; volume: "53(12):gkaf620. doi"; 
            pages: " 10.1093/nar/gkaf620"
COMMENT     Sequence Label: pGH916
FEATURES             Location/Qualifiers
     source          1..14959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2687..3865
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2918..3856
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3813..3833
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3872..3881
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4466..4489,4490..4492,4493..7048,7049..7051,
                     7052..8566)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(4466..4489,4490..4492,4493..8566)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8579..8827
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             8840..8860
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8867..8920
                     /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             8921..9619
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     misc_feature    9651..10239
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9704..9724)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10122..10133)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10242..10258)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10243..10259)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10764..10944
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10970..10987)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10976..11200
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11246..11674
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11333..11352)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11543..11564
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11683..11703)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11688..12017
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11868..12003
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11930..11949
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12065..12112
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12131..12505
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    12635..12756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12672..12691)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12726..12745
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12805..12821)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12805..12821)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12818..12840)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12829..12845
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(12853..12859,12860..12877,12878..12883))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12898..12919
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13036..13053)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13207..13795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13287..13306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13966..14757,14758..14826))
                     /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     14589..14608
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14827..14931)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta gctagctgca aagatggata aagttttaaa cagagaggaa
     2641 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg
     2701 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     2761 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     2821 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     2881 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
     2941 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
     3001 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
     3061 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
     3121 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
     3181 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
     3241 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
     3301 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
     3361 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
     3421 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
     3481 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     3541 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     3601 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
     3661 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     3721 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     3781 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     3841 ttttcttcca tttcaggtgt cgtgacgtac ggccaccatg gacagcctct tgatgaaccg
     3901 gagggagttt ctttaccaat tcaaaaatgt ccgctgggct aagggtcggc gtgagaccta
     3961 cctgtgctac gtagtgaaga ggcgtgacag tgctacatcc ttttcactgg actttggtta
     4021 tcttcgcaat aagaacggct gccacgtgga attgctcttc ctccgctaca tcacagactg
     4081 ggacctagac cctggccgct gctaccgcgt cacctggttc atctcctgga gcccctgcta
     4141 cgactgtgcc cgacatgtgg ccgactttct gcgagggaac cccaacatca gtctgaggat
     4201 cttcaccgcg cgcctctact tctgtgagga ccgcaaggct gagcccgagg ggctgcggcg
     4261 gctgcaccgc gccggggtgc aaatagccat catgaccttc aaagattatt tttactgctg
     4321 gaatactttt gtagaaaacc acggaagaac tttcgaagcc tgggaagggc tgcatgaaaa
     4381 ttcagttcgt ctctccagac agcttcggcg catccttagc ggcagcgaga ctcccgggac
     4441 ctcagagtcc gccacacccg aaagtgacaa gaagtacagc atcggcctgg ccatcggcac
     4501 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     4561 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     4621 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     4681 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     4741 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     4801 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     4861 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     4921 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     4981 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     5041 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     5101 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     5161 gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     5221 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     5281 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     5341 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     5401 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     5461 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     5521 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     5581 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     5641 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     5701 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     5761 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     5821 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     5881 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     5941 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     6001 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     6061 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     6121 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     6181 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     6241 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     6301 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     6361 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     6421 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     6481 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     6541 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     6601 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     6661 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     6721 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     6781 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     6841 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     6901 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     6961 gtccgactac gatgtggacc acatcgtgcc tcagagcttt ctgaaggacg actccatcga
     7021 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     7081 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     7141 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     7201 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     7261 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     7321 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     7381 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     7441 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     7501 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     7561 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     7621 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     7681 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     7741 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     7801 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     7861 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     7921 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     7981 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     8041 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     8101 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     8161 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     8221 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     8281 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     8341 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     8401 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     8461 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     8521 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacagcg gtggttctac
     8581 taatctgtca gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat
     8641 cctcatgctc ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact
     8701 cgtgcacacc gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc
     8761 ccctgaatac aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa
     8821 gatgctctct ggtggttctc ccaagaagaa gaggaaagtc ggatccgagg gcagaggaag
     8881 tctgctaaca tgcggtgacg tcgaggagaa tcctggccca agcgagctga ttaaggagaa
     8941 catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc
     9001 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg
     9061 cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac
     9121 cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt
     9181 cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac
     9241 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc
     9301 caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta
     9361 ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag
     9421 ccatctgatc gcaaacatca agaccacata tagatccaag aaacccgcta agaacctcaa
     9481 gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga
     9541 gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact
     9601 ggggcacaag cttaattagt aaggccgaat tcgatatcaa gcttatcggt aatcaacctc
     9661 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     9721 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     9781 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     9841 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     9901 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     9961 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
    10021 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
    10081 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
    10141 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
    10201 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag
    10261 acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg
    10321 cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt
    10381 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
    10441 gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc
    10501 acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc
    10561 cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag
    10621 ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc
    10681 cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc
    10741 gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc
    10801 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
    10861 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
    10921 agtcagtgtg gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg
    10981 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
    11041 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
    11101 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
    11161 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
    11221 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
    11281 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
    11341 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
    11401 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
    11461 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
    11521 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
    11581 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
    11641 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
    11701 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
    11761 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
    11821 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
    11881 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
    11941 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
    12001 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
    12061 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
    12121 gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
    12181 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
    12241 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
    12301 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
    12361 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
    12421 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
    12481 cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt
    12541 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
    12601 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
    12661 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
    12721 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
    12781 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
    12841 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
    12901 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
    12961 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
    13021 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
    13081 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
    13141 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
    13201 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
    13261 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
    13321 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
    13381 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
    13441 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
    13501 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
    13561 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
    13621 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
    13681 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
    13741 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
    13801 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
    13861 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
    13921 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
    13981 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
    14041 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
    14101 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
    14161 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
    14221 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
    14281 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
    14341 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
    14401 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
    14461 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
    14521 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
    14581 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
    14641 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
    14701 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
    14761 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
    14821 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
    14881 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
    14941 ccgaaaagtg ccacctgac
//
