LOCUS       sequence-271552-       11274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAEF5 plasmid with two gRNA inserted.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11274)
  AUTHORS   Agier N, Fleiss A, Delmas S, Fischer G
  TITLE     A Versatile Protocol to Generate Translocations in Yeast Genomes 
            Using CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2021;2196:181-198. doi: 
            10.1007/978-1-0716-0868-5_14.
  PUBMED    32889721
REFERENCE   2  (bases 1 to 11274)
  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: "Methods Mol
            Biol."; date: "2021"; volume: "2196:181-198. doi"; pages: " 
            10.1007/978-1-0716-0868-5_14"
COMMENT     Sequence Label: pAEF6
FEATURES             Location/Qualifiers
     source          1..11274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..65
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    182..203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(218..223,224..241,242..248)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    256..272
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     261..283
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     280..296
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     280..296
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     315..335
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        317..335
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        353..751
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     755..771
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     755..771
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             783..4886
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             783..4886
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             783..4886
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4899..4919
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4935..5182
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4951..4969)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        5204..5472
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5493..5567
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5572..5591
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        5614..5882
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5903..5977
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5982..6001
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(6023..6042)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6024..6042)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6052..6069)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6052..6068)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6061..6083)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            6173..7748
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        6173..6516
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             6517..7545
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             6517..7545
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      7551..7748
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     7956..7978
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8016..8034)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      8075..9417
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    8407..8454
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        9444..9548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9549..9617,9618..10409)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9767..9786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10580..11168
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11069..11088
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
       61 gcgaggaagc ggagagacgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
      121 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
      181 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
      241 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
      301 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctcatagctt
      361 caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc gcatcgccgt
      421 accacttcaa aacacccaag cacagcatac taaatttccc ctctttcttc ctctagggtg
      481 tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt
      541 cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa aatttttttt
      601 ttgatttttt tctctttcga tgacctccca ttgatattta agttaataaa cggtcttcaa
      661 tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgctca
      721 ttagaaagaa agcatagcaa tctaatctaa gttttctaga actagtggat cccccgggaa
      781 aaatggacaa gaagtactcc attgggctcg atatcggcac aaacagcgtc ggttgggccg
      841 tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc aataccgatc
      901 gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg gagacggccg
      961 aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag aatcggatct
     1021 gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct ttcttccata
     1081 ggctggagga gtcctttttg gtggaggagg ataaaaagca cgagcgccac ccaatctttg
     1141 gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat catctgagga
     1201 agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc gcgctggcgc
     1261 atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca gacaacagcg
     1321 atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc gaagagaacc
     1381 cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg tccaaatccc
     1441 ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc ctgtttggta
     1501 atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc gacctggccg
     1561 aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac aatctgctgg
     1621 cccagatcgg cgaccagtac gcagaccttt ttttggcggc aaagaacctg tcagacgcca
     1681 ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg ctgagcgcta
     1741 gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag gcccttgtca
     1801 gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa aatggctacg
     1861 ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt aagcccatct
     1921 tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa gatctgttgc
     1981 gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg ggcgaactgc
     2041 acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac agggaaaaga
     2101 ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc cggggaaatt
     2161 ccagattcgc gtggatgact cgcaaatcag aagagaccat cactccctgg aacttcgagg
     2221 aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact aactttgata
     2281 aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag tacttcacag
     2341 tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag ccagcattcc
     2401 tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac cggaaagtta
     2461 ccgtgaaaca gctcaaagaa gactatttca aaaagattga atgtttcgac tctgttgaaa
     2521 tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa
     2581 tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt gaggacattg
     2641 tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg aaaacttacg
     2701 ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat acaggatggg
     2761 ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga aagacaatcc
     2821 tggattttct taagtccgat ggatttgcca accggaactt catgcagttg atccatgatg
     2881 actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag ggggacagtc
     2941 ttcacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga atactgcaga
     3001 ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc gagaatatcg
     3061 ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac agtagggaaa
     3121 ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt aaggaacacc
     3181 cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg cagaacggca
     3241 gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac gacgtggatc
     3301 atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg ttgacaagat
     3361 ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc aagaaaatga
     3421 aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag ttcgataatc
     3481 tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc atcaaaaggc
     3541 agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat tcacgcatga
     3601 acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt actctgaagt
     3661 ctaagctggt ctcagatttc agaaaggact ttcagtttta taaggtgaga gagatcaaca
     3721 attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca cttatcaaaa
     3781 aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac gatgttagga
     3841 aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac ttcttttaca
     3901 gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag attcggaagc
     3961 gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag ggtagggatt
     4021 tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag
     4081 tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc gacaagctga
     4141 tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct cctacagtcg
     4201 cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg
     4261 tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg
     4321 actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt aagcttccca
     4381 agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc
     4441 tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg tatctggcca
     4501 gccactatga aaagctcaaa gggtctcccg aagataatga gcagaagcag ctgttcgtgg
     4561 aacaacacaa acactacctt gatgagatca tcgagcaaat aagcgaattc tccaaaagag
     4621 tgatcctcgc cgacgctaac ctcgataagg tgctttctgc ttacaataag cacagggata
     4681 agcccatcag ggagcaggca gaaaacatta tccacttgtt tactctgacc aacttgggcg
     4741 cgcctgcagc cttcaagtac ttcgacacca ccatagacag aaagcggtac acctctacaa
     4801 aggaggtcct ggacgccaca ctgattcatc agtcaattac ggggctctat gaaacaagaa
     4861 tcgacctctc tcagctcggt ggagacagca gggctgaccc caagaagaag aggaaggtgt
     4921 gatctcttct cgagtcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
     4981 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     5041 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     5101 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     5161 acgctcgaag gctttaattt gcggccggta ccgcgggccc gcttctttga aaagataatg
     5221 tatgattatg ctttcactca tatttataca gaaacttgat gttttctttc gagtatatac
     5281 aaggtgatta catgtacgtt tgaagtacaa ctctagattt tgtagtgccc tcttgggcta
     5341 gcggtaaagg tgcgcatttt ttcacaccct acaatgttct gttcaaaaga ttttggtcaa
     5401 acgctgtaga agtgaaagtt ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa
     5461 agataaatga tccatgtcga aagctacata tagttttaga gctagaaata gcaagttaaa
     5521 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt
     5581 tttttatgtc tgcggccgcg gtacccaatt cgctctttga aaagataatg tatgattatg
     5641 ctttcactca tatttataca gaaacttgat gttttctttc gagtatatac aaggtgatta
     5701 catgtacgtt tgaagtacaa ctctagattt tgtagtgccc tcttgggcta gcggtaaagg
     5761 tgcgcatttt ttcacaccct acaatgttct gttcaaaaga ttttggtcaa acgctgtaga
     5821 agtgaaagtt ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa agataaatga
     5881 tcgcaaaact aaaaaactgt atgttttaga gctagaaata gcaagttaaa ataaggctag
     5941 tccgttatca acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt tttttatgtc
     6001 tgcggccgcg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     6061 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     6121 acatcccaga tctgtttagc ttgcctcgtc cccgccgggt cacccggcca gcgacatgga
     6181 ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg
     6241 tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca tacattttga
     6301 tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cggacctgcg agcagggaaa
     6361 cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa
     6421 aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg
     6481 atacagttct cacatcacat ccgaacataa acaaccatgg gtaaaaagcc tgaactcacc
     6541 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     6601 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     6661 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     6721 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     6781 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     6841 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     6901 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     6961 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     7021 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     7081 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     7141 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     7201 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     7261 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     7321 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     7381 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     7441 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     7501 agtggaaacc gacgccccag cactcgtccg agggcaaagg aataatcagt actgacaata
     7561 aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc
     7621 tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag
     7681 atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg
     7741 ctatactgct gtcgattcga tactaacgcc gccatccagt gtcgaaaacg agctcgaatt
     7801 catcgatgat atcagatcca ctagtggcct atgcggccca atctgctctg atgccgcata
     7861 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     7921 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     7981 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     8041 ggttaatgtc atgataataa tggtttctta gtatgatcca atatcaaagg aaatgatagc
     8101 attgaaggat gagactaatc caattgagga gtggcagcat atagaacagc taaagggtag
     8161 tgctgaagga agcatacgat accccgcatg gaatgggata atatcacagg aggtactaga
     8221 ctacctttca tcctacataa atagacgcat ataagtacgc atttaagcat aaacacgcac
     8281 tatgccgttc ttctcatgta tatatatata caggcaacac gcagatatag gtgcgacgtg
     8341 aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg gaagcgctcg ttttcggaaa
     8401 cgctttgaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttcagagcg
     8461 cttttgaaaa ccaaaagcgc tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac
     8521 tgtaacgagc tactaaaata ttgcgaatac cgcttccaca aacattgctc aaaagtatct
     8581 ctttgctata tatctctgtg ctatatccct atataaccta cccatccacc tttcgctcct
     8641 tgaacttgca tctaaactcg acctctacat tttttatgtt tatctctagt attactcttt
     8701 agacaaaaaa attgtagtaa gaactattca tagagtgaat cgaaaacaat acgaaaatgt
     8761 aaacatttcc tatacgtagt atatagagac aaaatagaag aaaccgttca taattttctg
     8821 accaatgaag aatcatcaac gctatcactt tctgttcaca aagtatgcgc aatccacatc
     8881 ggtatagaat ataatcgggg atgcctttat cttgaaaaaa tgcacccgca gcttcgctag
     8941 taatcagtaa acgcgggaag tggagtcagg ctttttttat ggaagagaaa atagacacca
     9001 aagtagcctt cttctaacct taacggacct acagtgcaaa aagttatcaa gagactgcat
     9061 tatagagcgc acaaaggaga aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct
     9121 ctcgggatgc atttttgtag aacaaaaaag aagtatagat tctttgttgg taaaatagcg
     9181 ctctcgcgtt gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta
     9241 gcgctctcgc gttgcatttt tgttttacaa aaatgaagca cagattcttc gttggtaaaa
     9301 tagcgctttc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa
     9361 aattagcgct ctcgcgttgc atttttgttc tacaaaatga agcacagatg cttcgttcag
     9421 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     9481 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     9541 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     9601 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     9661 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     9721 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     9781 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     9841 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     9901 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     9961 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
    10021 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
    10081 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
    10141 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
    10201 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
    10261 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
    10321 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
    10381 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
    10441 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
    10501 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
    10561 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
    10621 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
    10681 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
    10741 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
    10801 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
    10861 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
    10921 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
    10981 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
    11041 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
    11101 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
    11161 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
    11221 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accg
//
