LOCUS       sequence-458308-       15352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ef1a-ABE7.10-T2A-tagBFP-wPRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15352)
  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 15352)
  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: pGH720
FEATURES             Location/Qualifiers
     source          1..15352
                     /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"
     CDS             join(5069..5092,5093..5095,5096..7651,7652..7654,
                     7655..9169)
                     /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(5069..5092,5093..5095,5096..9169)
                     /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             9224..9244
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9260..9313
                     /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             9314..10012
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     misc_feature    10044..10632
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10097..10117)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10515..10526)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10635..10651)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10636..10652)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11157..11337
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(11363..11380)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11369..11593
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11639..12067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11726..11745)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11936..11957
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12076..12096)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12081..12410
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      12261..12396
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12323..12342
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12458..12505
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12524..12898
                     /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    13028..13149
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13065..13084)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13119..13138
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13198..13214)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13198..13214)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13211..13233)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13222..13238
                     /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(13246..13252,13253..13270,13271..13276))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13291..13312
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13429..13446)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13600..14188)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13680..13699)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14359..15150,15151..15219))
                     /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     14982..15001
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15220..15324)
                     /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 tctgaggtgg aatttagcca
     3901 tgagtactgg atgcgccatg cgttgaccct tgcaaagaga gcctgggacg aaagagaagt
     3961 accggttggc gctgtacttg tgcataacaa ccgcgtaatt ggggaaggtt ggaatcgacc
     4021 aataggtcgc catgatccaa cagcacatgc agagattatg gcccttcggc agggcggcct
     4081 tgtaatgcag aactaccgcc ttattgacgc aacgctttac gtgacgcttg agccatgtgt
     4141 gatgtgtgcg ggtgcaatga tacactcaag gattggacgc gtggtatttg gggcaaggga
     4201 tgcaaaaaca ggagcggctg gatcactgat ggacgttttg catcaccctg gcatgaacca
     4261 ccgcgttgaa ataaccgagg gcatcctcgc cgacgagtgc gcggctcttc tgtcagactt
     4321 ctttaggatg cggcgacagg aaataaaagc acaaaagaag gcccagagca gcacagactc
     4381 cggcggctcc agcggtggta gctcaggatc cgagactccc ggcacttctg aaagcgcgac
     4441 gcctgagtcc tcaggagggt cctccggggg aagtagtgag gttgagttca gtcatgaata
     4501 ctggatgcgc catgcactga ccctggctaa acgggcgcgg gacgaacgag aagttccggt
     4561 gggggcggtg cttgtcctga acaaccgagt gattggtgaa ggttggaacc gggctatcgg
     4621 cttgcacgat ccgacggcgc acgccgaaat catggctctt cggcagggcg gtcttgtcat
     4681 gcagaattac aggctgattg acgcaacatt gtacgttaca tttgaacctt gtgtcatgtg
     4741 cgcaggcgca atgattcaca gcagaattgg gcgggtagtg tttggcgtaa gaaatgcaaa
     4801 gactggtgca gctggtagtc ttatggatgt gctgcattat ccgggtatga accaccgcgt
     4861 agaaatcacc gaaggcattc tcgcggatga atgcgctgcg ttgctttgtt atttctttcg
     4921 catgcccagg caggttttta atgcccaaaa gaaagcacaa tcaagtacag atagtggagg
     4981 atctagcggc ggcagctcag gttccgagac ccctgggact agcgaatctg ccacgcccga
     5041 gtcatccggc gggagttccg ggggctcaga caagaagtac agcatcggcc tggccatcgg
     5101 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     5161 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     5221 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     5281 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     5341 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     5401 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     5461 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     5521 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     5581 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     5641 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     5701 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     5761 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
     5821 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     5881 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     5941 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     6001 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     6061 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     6121 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     6181 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     6241 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     6301 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     6361 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     6421 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     6481 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     6541 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     6601 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     6661 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     6721 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     6781 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     6841 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     6901 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     6961 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     7021 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     7081 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     7141 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     7201 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     7261 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     7321 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     7381 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     7441 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     7501 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     7561 gctgtccgac tacgatgtgg accacatcgt gcctcagagc tttctgaagg acgactccat
     7621 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     7681 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     7741 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     7801 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     7861 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     7921 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     7981 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     8041 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     8101 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     8161 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     8221 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     8281 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     8341 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     8401 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     8461 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     8521 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     8581 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     8641 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     8701 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     8761 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     8821 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     8881 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     8941 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     9001 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     9061 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     9121 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca gcgctggagg
     9181 aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa
     9241 ggtggcggcc gctggatccg agggcagagg aagtctgcta acatgcggtg acgtcgagga
     9301 gaatcctggc ccaagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     9361 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     9421 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     9481 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     9541 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     9601 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     9661 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     9721 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     9781 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     9841 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     9901 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     9961 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt agtaaggccg
    10021 aattcgatat caagcttatc ggtaatcaac ctctggatta caaaatttgt gaaagattga
    10081 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
    10141 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
    10201 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
    10261 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
    10321 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
    10381 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
    10441 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
    10501 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
    10561 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
    10621 ccgcctcccc gcatcgatac cgtcgacctc gagacctaga aaaacatgga gcaatcacaa
    10681 gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg
    10741 aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg
    10801 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
    10861 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc
    10921 agaactacac accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc
    10981 tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt
    11041 tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt
    11101 ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt
    11161 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
    11221 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
    11281 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg
    11341 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
    11401 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
    11461 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
    11521 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
    11581 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
    11641 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
    11701 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
    11761 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
    11821 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
    11881 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
    11941 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
    12001 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
    12061 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
    12121 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
    12181 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
    12241 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
    12301 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
    12361 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
    12421 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
    12481 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
    12541 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
    12601 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
    12661 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
    12721 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
    12781 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
    12841 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
    12901 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
    12961 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
    13021 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
    13081 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
    13141 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
    13201 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
    13261 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
    13321 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
    13381 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
    13441 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
    13501 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
    13561 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
    13621 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
    13681 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
    13741 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
    13801 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
    13861 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
    13921 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
    13981 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
    14041 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
    14101 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
    14161 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
    14221 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
    14281 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
    14341 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
    14401 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
    14461 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
    14521 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
    14581 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
    14641 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
    14701 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
    14761 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
    14821 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
    14881 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
    14941 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
    15001 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
    15061 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
    15121 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
    15181 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
    15241 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
    15301 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ac
//
