LOCUS       sequence-266848-       10036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of  BE4am-Cas9 Base-editor from an EF1a promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10036)
  AUTHORS   Standage-Beier K, Tekel SJ, Brookhouser N, Schwarz G, Nguyen T, Wang
            X, Brafman DA
  TITLE     A transient reporter for editing enrichment (TREE) in human cells.
  JOURNAL   Nucleic Acids Res. 2019 Aug 20. pii: 5552066. doi: 
            10.1093/nar/gkz713.
  PUBMED    31428784
REFERENCE   2  (bases 1 to 10036)
  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. 2019 Aug 20. pii: 5552066. doi: 10.1093/nar/gkz713."
COMMENT     Sequence Label: pEF-BE4Gam
FEATURES             Location/Qualifiers
     source          1..10036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        12..1194
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          243..1185
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1142..1162
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1204..1223
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1204..1222
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1230..1239
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1806..2489
                     /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="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             join(2586..5984,5985..5987,5988..6584,6585,6586..6588,
                     6589..6590,6591..6593,6594..6686)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /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(2586..2609,2610..2612,2613..6686)
                     /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             6717..6965
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6996..7244
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             7257..7277
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7286..7303
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7326..7343)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7332..7556
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7627..7643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7627..7643)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7640..7662)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7651..7667
                     /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(7675..7681,7682..7699,7700..7705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7720..7741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7858..7875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8029..8617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8109..8128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8788..9579,9580..9648))
                     /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     9411..9430
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9649..9753)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9832..9851)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 actagtcgtg aggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
       61 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
      121 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
      181 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
      241 aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg
      301 tgccttgaat tacttccacg cccctggctg cagtacgtga ttcttgatcc cgagcttcgg
      361 gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct
      421 tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc
      481 gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg
      541 acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt
      601 tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg
      661 aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg
      721 cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg
      781 gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg
      841 agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg
      901 aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg
      961 tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag
     1021 gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct
     1081 tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt
     1141 ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaggcggc
     1201 cgctaatacg actcactata gggagagccg ccaccatggc taaaccagca aaacgtatca
     1261 agagtgccgc agcggcttat gtgccacaaa accgcgatgc ggtgattacc gatattaaac
     1321 gcatcgggga tttacagcgc gaagcatcac gtctggaaac ggaaatgaat gatgccatcg
     1381 cggaaattac ggagaaattt gcggcccgga ttgcaccgat taaaaccgat attgaaaccc
     1441 tttcaaaagg cgttcaggga tggtgtgaag cgaaccgcga cgaactgacg aacggcggca
     1501 aagtgaagac ggcgaatctt gtcaccggtg atgtatcgtg gcgggtccgt ccaccatcag
     1561 taagtattcg tggtatggat gcagtgatgg aaacgctgga gcgtcttggc ctgcaacgct
     1621 ttattcgcac gaagcaggaa atcaacaagg aagcgatttt actggaaccg aaagcggtcg
     1681 caggcgttgc cggaattaca gttaaatcag gcattgagga tttttctatt attccatttg
     1741 aacaggaagc cggtattagc ggcagcgaga ctcccgggac ctcagagtcc gctacacccg
     1801 aaagtagctc agagactggc ccagtggctg tggaccccac attgagacgg cggatcgagc
     1861 cccatgagtt tgaggtattc ttcgatccga gagagctccg caaggagacc tgcctgcttt
     1921 acgaaattaa ttgggggggc cggcactcca tttggcgaca tacatcacag aacactaaca
     1981 agcacgtcga agtcaacttc atcgagaagt tcacgacaga aagatatttc tgtccgaaca
     2041 caaggtgcag cattacctgg tttctcagct ggagcccatg cggcgaatgt agtagggcca
     2101 tcactgaatt cctgtcaagg tatccccacg tcactctgtt tatttacatc gcaaggctgt
     2161 accaccacgc tgacccccgc aatcgacaag gcctgcggga tttgatctct tcaggtgtga
     2221 ctatccaaat tatgactgag caggagtcag gatactgctg gagaaacttt gtgaattata
     2281 gcccgagtaa tgaagcccac tggcctaggt atccccatct gtgggtacga ctgtacgttc
     2341 ttgaactgta ctgcatcata ctgggcctgc ctccttgtct caacattctg agaaggaagc
     2401 agccacagct gacattcttt accatcgctc ttcagtcttg tcattaccag cgactgcccc
     2461 cacacattct ctgggccacc gggttgaaat ctggtggttc ttctggtggt tctagcggca
     2521 gcgagactcc cgggacctca gagtccgcca cacccgaaag ttctggtggt tcttctggtg
     2581 gttctgataa aaagtattct attggtttag ccatcggcac taattccgtt ggatgggctg
     2641 tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg aacacagacc
     2701 gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc gaaacggcag
     2761 aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag aaccgaatat
     2821 gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct ttctttcacc
     2881 gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac cccatctttg
     2941 gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat cacctcagaa
     3001 aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg gctcttgccc
     3061 atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg gacaactcgg
     3121 atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt gaagagaacc
     3181 ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc tctaaatccc
     3241 gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg ttgttcggta
     3301 accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc gacttagctg
     3361 aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac aatctactgg
     3421 cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt agcgatgcaa
     3481 tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg ttatccgctt
     3541 caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag gccctagtcc
     3601 gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa aacgggtacg
     3661 caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc aaacccatat
     3721 tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa gatctactgc
     3781 gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta ggcgaattgc
     3841 atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat cgtgaaaaga
     3901 ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc cgagggaact
     3961 ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccatgg aattttgagg
     4021 aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc aactttgaca
     4081 agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag tatttcacag
     4141 tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa cccgcctttc
     4201 taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac cgcaaagtga
     4261 cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat tctgtcgaga
     4321 tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac ctcctaaaga
     4381 taattaaaga taaggacttc ctggataacg aagagaatga agatatctta gaagatatag
     4441 tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta aaaacatacg
     4501 ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat acgggctggg
     4561 gacgattgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt aaaactattc
     4621 tcgattttct aaagagcgac ggcttcgcca ataggaactt tatgcagctg atccatgatg
     4681 actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa ggggactcat
     4741 tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc atactccaga
     4801 cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg gaaaacattg
     4861 taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac agtcgagagc
     4921 ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta aaggagcatc
     4981 ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta caaaatggaa
     5041 gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac gacgtcgatc
     5101 acattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg cttacacgct
     5161 cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta aagaaaatga
     5221 agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag ttcgataact
     5281 taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt attaaacgtc
     5341 agctcgtgga aacccgccaa atcacaaagc atgttgcaca gatactagat tcccgaatga
     5401 atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc actttaaagt
     5461 caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg gagataaata
     5521 actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca ctcattaaga
     5581 aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat gacgtccgta
     5641 agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac ttcttttatt
     5701 ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag atacgcaaac
     5761 gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag ggccgggact
     5821 tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag aaaactgagg
     5881 tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt gataagctca
     5941 tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgatagc cctacagttg
     6001 cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa ctgaagtcag
     6061 tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag aaccccatcg
     6121 acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt aaactaccaa
     6181 agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc gccggagagc
     6241 ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg tatttagcgt
     6301 cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa ctttttgttg
     6361 agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc agtaagagag
     6421 tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag cacagggata
     6481 aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc aacctcggcg
     6541 ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac acttctacca
     6601 aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat gaaactcgga
     6661 tagatttgtc acagcttggg ggtgactctg gtggttctgg aggatctggt ggttctacta
     6721 atctgtcaga tattattgaa aaggagaccg gtaagcaact ggttatccag gaatccatcc
     6781 tcatgctccc agaggaggtg gaagaagtca ttgggaacaa gccggaaagc gatatactcg
     6841 tgcacaccgc ctacgacgag agcaccgacg agaatgtcat gcttctgact agcgacgccc
     6901 ctgaatacaa gccttgggct ctggtcatac aggatagcaa cggtgagaac aagattaaga
     6961 tgctctctgg tggttctgga ggatctggtg gttctactaa tctgtcagat attattgaaa
     7021 aggagaccgg taagcaactg gttatccagg aatccatcct catgctccca gaggaggtgg
     7081 aagaagtcat tgggaacaag ccggaaagcg atatactcgt gcacaccgcc tacgacgaga
     7141 gcaccgacga gaatgtcatg cttctgacta gcgacgcccc tgaatacaag ccttgggctc
     7201 tggtcataca ggatagcaac ggtgagaaca agattaagat gctctctggt ggttctccca
     7261 agaagaagag gaaagtctaa ccggtcatca tcaccatcac cattgagttt aaacccgctg
     7321 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     7381 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     7441 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     7501 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     7561 tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga gcttggcgta
     7621 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     7681 acgagccgga agcataaagt gtaaagccta gggtgcctaa tgagtgagct aactcacatt
     7741 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7801 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     7861 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7921 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7981 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     8041 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     8101 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     8161 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     8221 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     8281 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     8341 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     8401 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     8461 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     8521 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     8581 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     8641 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     8701 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     8761 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     8821 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     8881 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     8941 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     9001 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     9061 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     9121 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     9181 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     9241 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     9301 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     9361 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     9421 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     9481 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     9541 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     9601 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     9661 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     9721 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     9781 cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca gtacaatctg
     9841 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     9901 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     9961 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
    10021 ttgacattga ttattg
//
