LOCUS       sequence-458361-       14799 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Ef1a-BE3-T2A-BlastR-wPRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14799)
  AUTHORS   Schwartz CI, Abell NS, Li A, Aradhana, Tycko J, Truong A, Montgomery
            SB, Hess GT
  TITLE     Towards optimizing diversifying base editors for high-throughput 
            studies of single- nucleotide variants.
  JOURNAL   bioRxiv [Preprint]. 2024 Nov 19:2024.11.18.621003. doi: 
            10.1101/2024.11.18.621003.
  PUBMED    39605325
REFERENCE   2  (bases 1 to 14799)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Nov 19:2024.11.18.621003. doi: 
            10.1101/2024.11.18.621003."
COMMENT     Sequence Label: pGH911
FEATURES             Location/Qualifiers
     source          1..14799
                     /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             3878..4564
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             join(4613..4636,4637..4639,4640..7195,7196..7198,
                     7199..8713)
                     /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(4613..4636,4637..4639,4640..8713)
                     /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             8726..8974
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             8987..9007
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9014..9067
                     /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             9068..9466
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    9491..10079
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9544..9564)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9962..9973)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10082..10098)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10083..10099)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10604..10784
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10810..10827)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10816..11040
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11086..11514
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11173..11192)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11383..11404
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11523..11543)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11528..11857
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11708..11843
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11770..11789
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11905..11952
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11971..12345
                     /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    12475..12596
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12512..12531)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12566..12585
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12645..12661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12645..12661)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12658..12680)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12669..12685
                     /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(12693..12699,12700..12717,12718..12723))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12738..12759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12876..12893)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13047..13635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13127..13146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13806..14597,14598..14666))
                     /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     14429..14448
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14667..14771)
                     /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 agctcagaga ctggcccagt
     3901 ggctgtggac cccacattga gacggcggat cgagccccat gagtttgagg tattcttcga
     3961 tccgagagag ctccgcaagg agacctgcct gctttacgaa attaattggg ggggccggca
     4021 ctccatttgg cgacatacat cacagaacac taacaagcac gtcgaagtca acttcatcga
     4081 gaagttcacg acagaaagat atttctgtcc gaacacaagg tgcagcatta cctggtttct
     4141 cagctggagc ccatgcggcg aatgtagtag ggccatcact gaattcctgt caaggtatcc
     4201 ccacgtcact ctgtttattt acatcgcaag gctgtaccac cacgctgacc cccgcaatcg
     4261 acaaggcctg cgggatttga tctcttcagg tgtgactatc caaattatga ctgagcagga
     4321 gtcaggatac tgctggagaa actttgtgaa ttatagcccg agtaatgaag cccactggcc
     4381 taggtatccc catctgtggg tacgactgta cgttcttgaa ctgtactgca tcatactggg
     4441 cctgcctcct tgtctcaaca ttctgagaag gaagcagcca cagctgacat tctttaccat
     4501 cgctcttcag tcttgtcatt accagcgact gcccccacac attctctggg ccaccgggtt
     4561 gaaaagcggc agcgagactc ccgggacctc agagtccgcc acacccgaaa gtgacaagaa
     4621 gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     4681 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     4741 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     4801 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     4861 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     4921 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     4981 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     5041 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     5101 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     5161 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     5221 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     5281 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct
     5341 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     5401 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     5461 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     5521 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     5581 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     5641 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     5701 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     5761 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     5821 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     5881 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     5941 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     6001 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     6061 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     6121 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     6181 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     6241 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     6301 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     6361 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     6421 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     6481 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     6541 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     6601 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     6661 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     6721 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     6781 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     6841 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     6901 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     6961 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     7021 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     7081 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccaca tcgtgcctca
     7141 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     7201 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     7261 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     7321 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     7381 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     7441 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     7501 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     7561 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     7621 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     7681 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     7741 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     7801 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     7861 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     7921 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     7981 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     8041 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     8101 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     8161 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     8221 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     8281 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     8341 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     8401 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     8461 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     8521 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     8581 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     8641 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     8701 gctgggaggc gacagcggtg gttctactaa tctgtcagat attattgaaa aggagaccgg
     8761 taagcaactg gttatccagg aatccatcct catgctccca gaggaggtgg aagaagtcat
     8821 tgggaacaag ccggaaagcg atatactcgt gcacaccgcc tacgacgaga gcaccgacga
     8881 gaatgtcatg cttctgacta gcgacgcccc tgaatacaag ccttgggctc tggtcataca
     8941 ggatagcaac ggtgagaaca agattaagat gctctctggt ggttctccca agaagaagag
     9001 gaaagtcgga tccgagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc
     9061 tggcccaatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc
     9121 tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag
     9181 cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga
     9241 actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc
     9301 gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct
     9361 cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt
     9421 tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagaat tcgatatcaa
     9481 gcttatcggt aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     9541 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     9601 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     9661 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     9721 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     9781 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     9841 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     9901 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     9961 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
    10021 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
    10081 tcgataccgt cgacctcgag acctagaaaa acatggagca atcacaagta gcaatacagc
    10141 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc
    10201 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
    10261 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat
    10321 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc
    10381 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
    10441 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag
    10501 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct
    10561 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga
    10621 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
    10681 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
    10741 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc
    10801 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
    10861 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
    10921 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
    10981 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
    11041 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
    11101 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
    11161 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
    11221 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
    11281 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
    11341 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
    11401 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
    11461 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
    11521 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
    11581 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
    11641 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
    11701 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
    11761 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
    11821 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
    11881 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
    11941 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
    12001 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
    12061 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
    12121 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
    12181 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
    12241 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
    12301 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
    12361 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
    12421 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
    12481 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
    12541 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
    12601 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
    12661 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
    12721 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
    12781 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
    12841 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
    12901 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
    12961 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
    13021 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
    13081 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
    13141 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
    13201 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
    13261 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
    13321 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
    13381 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
    13441 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
    13501 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
    13561 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
    13621 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
    13681 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
    13741 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
    13801 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
    13861 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
    13921 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
    13981 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
    14041 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
    14101 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
    14161 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
    14221 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
    14281 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
    14341 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
    14401 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
    14461 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
    14521 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
    14581 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
    14641 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
    14701 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
    14761 ggggttccgc gcacatttcc ccgaaaagtg ccacctgac
//
