LOCUS       sequence-384200-       13143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  [E*](104) construct. Theophylline inducible Cas9 (S. pyogenes) 
            expression. Two BsaI-sites allow for simultaneous insertion of sgRNA
            and donor DNA. Includes a nickel-inducible curing system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13143)
  AUTHORS   Cengic I, Canadas IC, Minton NP, Hudson EP
  TITLE     Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated 
            Single-Target Genome Editing in Synechocystis Sp. PCC 6803.
  JOURNAL   ACS Synth Biol. 2022 Aug 15. doi: 10.1021/acssynbio.2c00375.
  PUBMED    35969224
REFERENCE   2  (bases 1 to 13143)
  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: "ACS Synth 
            Biol. 2022 Aug 15. doi: 10.1021/acssynbio.2c00375."
COMMENT     Sequence Label: 
            pPMQAK1-Ptrc-[E*](104)-Cas9-B0015-lacZ-PnrsB-mazF-LVA
FEATURES             Location/Qualifiers
     source          1..13143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    62..78
                     /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)."
     CDS             160..4266
                     /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             160..4263
                     /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"
     terminator      4278..4349
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4365..4392
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4512..4835)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     4517..4536
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4730..4752
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4744..4761
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4745..4761
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4762..4818
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4831..4847)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4831..4847)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    4855..4871
                     /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(4879..4885,4886..4903,4904..4909))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4924..4945
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             5450..5482
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag (LVA)
                     /note="mutant LVA variant that confers accelerated 
                     degradation under some conditions (Andersen et al., 1998)"
                     /translation="AANDENYALVA"
     terminator      5490..5547
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(5571..5588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5729..6123
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     oriT            6465..6552
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     CDS             7791..8762
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     CDS             9273..10112
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             10099..10950
                     /codon_start=1
                     /gene="repC"
                     /product="replication protein C of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepC
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     CDS             complement(11073..11888)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(11073..11888)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     11796..11815
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     11880..11899
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(join(12043..12834,12835..12903))
                     /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     12666..12685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12904..13008)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     13076..13094
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(13098..13141)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 tgccaaatat tctgaaatga gctgttgaca attaatcatc cggctcgtat tgtgtgtgga
       61 attgtgagcg gataacaatt tcatacgctc acaattggta ccggtgatac cagcatcgtc
      121 ttgatgccct tggcagcacc ctgctaagga ggcaacaaga tggataagaa atactcaata
      181 ggcttagata tcggcacaaa tagcgtcgga tgggcggtga tcactgatga atataaggtt
      241 ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc acagtatcaa aaaaaatctt
      301 ataggggctc ttttatttga cagtggagag acagcggaag cgactcgtct caaacggaca
      361 gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt atctacagga gattttttca
      421 aatgagatgg cgaaagtaga tgatagtttc tttcatcgac ttgaagagtc ttttttggtg
      481 gaagaagata agaagcatga acgtcatcct atttttggaa atatagtaga tgaagttgct
      541 tatcatgaga aatatccaac tatctatcat ctgcgaaaaa aattggtaga ttctactgat
      601 aaagcggatt tgcgcttaat ctatttggcc ttagcgcata tgattaagtt tcgtggtcat
      661 tttttgattg agggagattt aaatcctgat aatagtgatg tggacaaact atttatccag
      721 ttggtacaaa cctacaatca attatttgaa gaaaacccta ttaacgcaag tggagtagat
      781 gctaaagcga ttctttctgc acgattgagt aaatcaagac gattagaaaa tctcattgct
      841 cagctccccg gtgagaagaa aaatggctta tttgggaatc tcattgcttt gtcattgggt
      901 ttgaccccta attttaaatc aaattttgat ttggcagaag atgctaaatt acagctttca
      961 aaagatactt acgatgatga tttagataat ttattggcgc aaattggaga tcaatatgct
     1021 gatttgtttt tggcagctaa gaatttatca gatgctattt tactttcaga tatcctaaga
     1081 gtaaatactg aaataactaa ggctccccta tcagcttcaa tgattaaacg ctacgatgaa
     1141 catcatcaag acttgactct tttaaaagct ttagttcgac aacaacttcc agaaaagtat
     1201 aaagaaatct tttttgatca atcaaaaaac ggatatgcag gttatattga tgggggagct
     1261 agccaagaag aattttataa atttatcaaa ccaattttag aaaaaatgga tggtactgag
     1321 gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca agcaacggac ctttgacaac
     1381 ggctctattc cccatcaaat tcacttgggt gagctgcatg ctattttgag aaggcaagag
     1441 gacttttatc catttttaaa agacaatcgt gagaagattg aaaaaatctt gacttttcgc
     1501 attccttatt atgttggtcc attggcgcgt ggcaatagtc gttttgcatg gatgactcgg
     1561 aagtctgaag aaacaattac cccatggaat tttgaagaag ttgtcgataa aggtgcttca
     1621 gctcaatcat ttattgaacg catgacaaac tttgataaaa atcttccaaa tgaaaaagta
     1681 ctaccaaaac atagtttgct ttatgagtat tttacggttt ataacgaatt gacaaaggtc
     1741 aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc
     1801 attgttgatt tactcttcaa aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat
     1861 tatttcaaaa aaatagaatg ttttgatagt gttgaaattt caggagttga agatagattt
     1921 aatgcttcat taggtaccta ccatgatttg ctaaaaatta ttaaagataa agattttttg
     1981 gataatgaag aaaatgaaga tatcttagag gatattgttt taacattgac cttatttgaa
     2041 gatagggaga tgattgagga aagacttaaa acatatgctc acctctttga tgataaggtg
     2101 atgaaacagc ttaaacgtcg ccgttatact ggttggggac gtttgtctcg aaaattgatt
     2161 aatggtatta gggataagca atctggcaaa acaatattag attttttgaa atcagatggt
     2221 tttgccaatc gcaattttat gcagctgatc catgatgata gtttgacatt taaagaagat
     2281 attcaaaaag cacaagtgtc tggacaaggc gatagtttac atgaacatat tgcaaattta
     2341 gctggtagcc ctgctattaa aaaaggtatt ttacagactg taaaagttgt tgatgaattg
     2401 gtcaaagtaa tggggcggca taagccagaa aatatcgtta ttgaaatggc acgtgaaaat
     2461 cagacaactc aaaagggcca gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt
     2521 atcaaagaat taggaagtca gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa
     2581 aatgaaaagc tctatctcta ttatctccaa aatggaagag acatgtatgt ggaccaagaa
     2641 ttagatatta atcgtttaag tgattatgat gtcgatcaca ttgttccaca aagtttcctt
     2701 aaagacgatt caatagacaa taaggtctta acgcgttctg ataaaaatcg tggtaaatcg
     2761 gataacgttc caagtgaaga agtagtcaaa aagatgaaaa actattggag acaacttcta
     2821 aacgccaagt taatcactca acgtaagttt gataatttaa cgaaagctga acgtggaggt
     2881 ttgagtgaac ttgataaagc tggttttatc aaacgccaat tggttgaaac tcgccaaatc
     2941 actaagcatg tggcacaaat tttggatagt cgcatgaata ctaaatacga tgaaaatgat
     3001 aaacttattc gagaggttaa agtgattacc ttaaaatcta aattagtttc tgacttccga
     3061 aaagatttcc aattctataa agtacgtgag attaacaatt accatcatgc ccatgatgcg
     3121 tatctaaatg ccgtcgttgg aactgctttg attaagaaat atccaaaact tgaatcggag
     3181 tttgtctatg gtgattataa agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa
     3241 gaaataggca aagcaaccgc aaaatatttc ttttactcta atatcatgaa cttcttcaaa
     3301 acagaaatta cacttgcaaa tggagagatt cgcaaacgcc ctctaatcga aactaatggg
     3361 gaaactggag aaattgtctg ggataaaggg cgagattttg ccacagtgcg caaagtattg
     3421 tccatgcccc aagtcaatat tgtcaagaaa acagaagtac agacaggcgg attctccaag
     3481 gagtcaattt taccaaaaag aaattcggac aagcttattg ctcgtaaaaa agactgggat
     3541 ccaaaaaaat atggtggttt tgatagtcca acggtagctt attcagtcct agtggttgct
     3601 aaggtggaaa aagggaaatc gaagaagtta aaatccgtta aagagttact agggatcaca
     3661 attatggaaa gaagttcctt tgaaaaaaat ccgattgact ttttagaagc taaaggatat
     3721 aaggaagtta aaaaagactt aatcattaaa ctacctaaat atagtctttt tgagttagaa
     3781 aacggtcgta aacggatgct ggctagtgcc ggagaattac aaaaaggaaa tgagctggct
     3841 ctgccaagca aatatgtgaa ttttttatat ttagctagtc attatgaaaa gttgaagggt
     3901 agtccagaag ataacgaaca aaaacaattg tttgtggagc agcataagca ttatttagat
     3961 gagattattg agcaaatcag tgaattttct aagcgtgtta ttttagcaga tgccaattta
     4021 gataaagttc ttagtgcata taacaaacat agagacaaac caatacgtga acaagcagaa
     4081 aatattattc atttatttac gttgacgaat cttggagctc ccgctgcttt taaatatttt
     4141 gatacaacaa ttgatcgtaa acgatatacg tctacaaaag aagttttaga tgccactctt
     4201 atccatcaat ccatcactgg tctttatgaa acacgcattg atttgagtca gctaggaggt
     4261 gactaataac caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     4321 ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg
     4381 ggcctttctg cgtttatata ctagaggttt ggagaccacg tgttcacagc ttgtctgtaa
     4441 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4501 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     4561 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     4621 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     4681 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     4741 cgttgtaaaa cgacggccag tgaattcgag ctcggtaccc ggggatcctc tagagtcgac
     4801 ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4861 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4921 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4981 aaacctgtcg tgccagctgc acacgtggtc tcgtggcaca gtctgatctt agcgggggaa
     5041 ggagattttc acctgaattt cataccccct ttggcagact gggaaaatct tggacaaatt
     5101 cccaatttga ggtggtatgg taagccgata cgtacccgat atgggcgatc tgatttgggt
     5161 tgattttgac ccgacaaaag gtagcgagca agctggacat cgtccagctg ttgtcctgag
     5221 tcctttcatg tacaacaaca aaacaggtat gtgtctgtgt gttccttgta caacgcaatc
     5281 aaaaggatat ccgttcgaag ttgttttatc cggtcaggaa cgtgatggcg tagcgttagc
     5341 tgatcaggta aaaagtatcg cctggcgggc aagaggagca acgaagaaag gaacagttgc
     5401 cccagaggaa ttacaactca ttaaagccaa aattaacgta ctgattgggg ctgcaaacga
     5461 cgaaaactac gctttagtag cttaggggat ccggcaaaaa agggcaaggt gtcaccaccc
     5521 tgcccttttt ctttaaaacc gaaaagatta cttcgcgtta tgcaggcttc ctcgctcact
     5581 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5641 atcaattgag ttcttttacc ctcagccgaa atgcctgccg ttgctagaca ttgccagcca
     5701 gtgcccgtca ctcccgtact aactgtcacg aacccctgca ataactgtca cgcccccctg
     5761 caataactgt cacgaacccc tgcaataact gtcacgcccc caaacctgca aacccagcag
     5821 gggcgggggc tggcggggtg ttggaaaaat ccatccatga ttatctaaga ataatccact
     5881 aggcgcggtt atcagcgccc ttgtggggcg ctgctgccct tgcccaatat gcccggccag
     5941 aggccggata gctggtctat tcgctgcgct aggctacaca ccgccccacc gctgcgcggc
     6001 agggggaaag gcgggcaaag cccgctaaac cccacaccaa accccgcaga aatacgctgg
     6061 agcgctttta gccgctttag cggcctttcc ccctacccga agggtggggg cgcgtgtgca
     6121 gccccgcagg gcctgtctcg gtcgatcatt cagcccggct catccttctg gcgtggcggc
     6181 agaccgaaca aggcgcggtc gtggtcgcgt tcaaggtacg catccattgc cgccatgagc
     6241 cgatcctccg gccactcgct gctgttcacc ttggccaaaa tcatggcccc caccagcacc
     6301 ttgcgccttg tttcgttctt gcgctcttgc tgctgttccc ttgcccgcac ccgctgaatt
     6361 tcggcattga ttcgcgctcg ttgttcttcg agcttggcca gccgatccgc cgccttgttg
     6421 ctccccttaa ccatcttgac accccattgt taatgtgctg tctcgtaggc tatcatggag
     6481 gcacagcggc ggcaatcccg accctacttt gtaggggagg gcgcacttac cggtttctct
     6541 tcgagaaact ggcctaacgg ccacccttcg ggcggtgcgc tctccgaggg ccattgcatg
     6601 gagccgaaaa gcaaaagcaa cagcgaggca gcatggcgat ttatcacctt acggcgaaaa
     6661 ccggcagcag gtcgggcggc caatcggcca gggccaaggc cgactacatc cagcgcgaag
     6721 gcaagtatgc ccgcgacatg gatgaagtct tgcacgccga atccgggcac atgccggagt
     6781 tcgtcgagcg gcccgccgac tactgggatg ctgccgacct gtatgaacgc gccaatgggc
     6841 ggctgttcaa ggaggtcgaa tttgccctgc cggtcgagct gaccctcgac cagcagaagg
     6901 cgctggcgtc cgagttcgcc cagcacctga ccggtgccga gcgcctgccg tatacgctgg
     6961 ccatccatgc cggtggcggc gagaacccgc actgccacct gatgatctcc gagcggatca
     7021 atgacggcat cgagcggccc gccgctcagt ggttcaagcg gtacaacggc aagaccccgg
     7081 agaagggcgg ggcacagaag aacgaagcgc tcaagcccaa ggcatggctt gagcagaccc
     7141 gcgaggcatg ggccgaccat gccaaccggg cattagagcg ggctggccac gacgcccgca
     7201 ttgaccacag aacacttgag gcgcagggca tcgagcgcct gcccggtgtt cacctggggc
     7261 cgaacgtggt ggagatggaa ggccggggca tccgcaccga ccgggcagac gtggccctga
     7321 acatcgacac cgccaacgcc cagatcatcg acttacagga ataccgggag gcaatagacc
     7381 atgaacgcaa tcgacagagt gaagaaatcc agaggcatca acgagttagc ggagcagatc
     7441 gaaccgctgg cccagagcat ggcgacactg gccgacgaag cccggcaggt catgagccag
     7501 acccagcagg ccagcgaggc gcaggcggcg gagtggctga aagcccagcg ccagacaggg
     7561 gcggcatggg tggagctggc caaagagttg cgggaggtag ccgccgaggt gagcagcgcc
     7621 gcgcagagcg cccggagcgc gtcgcggggg tggcactgga agctatggct aaccgtgatg
     7681 ctggcttcca tgatgcctac ggtggtgctg ctgatcgcat cgttgctctt gctcgacctg
     7741 acgccactga caaccgagga cggctcgatc tggctgcgct tggtggcccg atgaagaacg
     7801 acaggacttt gcaggccata ggccgacagc tcaaggccat gggctgtgag cgcttcgata
     7861 tcggcgtcag ggacgccacc accggccaga tgatgaaccg ggaatggtca gccgccgaag
     7921 tgctccagaa cacgccatgg ctcaagcgga tgaatgccca gggcaatgac gtgtatatca
     7981 ggcccgccga gcaggagcgg catggtctgg tgctggtgga cgacctcagc gagtttgacc
     8041 tggatgacat gaaagccgag ggccgggagc ctgccctggt agtggaaacc agcccgaaga
     8101 actatcaggc atgggtcaag gtggccgacg ccgcaggcgg tgaacttcgg gggcagattg
     8161 cccggacgct ggccagcgag tacgacgccg acccggccag cgccgacagc cgccactatg
     8221 gccgcttggc gggcttcacc aaccgcaagg acaagcacac cacccgcgcc ggttatcagc
     8281 cgtgggtgct gctgcgtgaa tccaagggca agaccgccac cgctggcccg gcgctggtgc
     8341 agcaggctgg ccagcagatc gagcaggccc agcggcagca ggagaaggcc cgcaggctgg
     8401 ccagcctcga actgcccgag cggcagctta gccgccaccg gcgcacggcg ctggacgagt
     8461 accgcagcga gatggccggg ctggtcaagc gcttcggtga tgacctcagc aagtgcgact
     8521 ttatcgccgc gcagaagctg gccagccggg gccgcagtgc cgaggaaatc ggcaaggcca
     8581 tggccgaggc cagcccagcg ctggcagagc gcaagcccgg ccacgaagcg gattacatcg
     8641 agcgcaccgt cagcaaggtc atgggtctgc ccagcgtcca gcttgcgcgg gccgagctgg
     8701 cacgggcacc ggcaccccgc cagcgaggca tggacagggg cgggccagat ttcagcatgt
     8761 agtgcttgcg ttggtactca cgcctgttat actatgagta ctcacgcaca gaagggggtt
     8821 ttatggaata cgaaaaaagc gcttcagggt cggtctacct gatcaaaagt gacaagggct
     8881 attggttgcc cggtggcttt ggttatacgt caaacaaggc cgaggctggc cgcttttcag
     8941 tcgctgatat ggccagcctt aaccttgacg gctgcacctt gtccttgttc cgcgaacaca
     9001 agcctttcgg ccccggcaag tttctcggtg actgatatga aagaccaaaa ggacaagcag
     9061 accggcgacc tgctggccag ccctgacgct gtacgccaag cgcgatatgc cgagcgcatg
     9121 aaggccaaag ggatgcgtca gcgcaagttc tggctgaccg acgacgaata cgaggcgctg
     9181 cgcgagtgcc tggaagaact cagagcggcg cagggcgggg gtagtgaccc cgccagcgcc
     9241 taaccaccaa ctgcctgcaa aggaggcaat caatggctac ccataagcct atcaatattc
     9301 tggaggcgtt cgcagcagcg ccgccaccgc tggactacgt tttgcccaac atggtggccg
     9361 gtacggtcgg ggcgctggtg tcgcccggtg gtgccggtaa atccatgctg gccctgcaac
     9421 tggccgcaca gattgcaggc gggccggatc tgctggaggt gggcgaactg cccaccggcc
     9481 cggtgatcta cctgcccgcc gaagatccgc ccaccgccat tcatcaccgc ctgcacgccc
     9541 ttggggcgca cctcagcgcc gaggaacggc aagccgtggc tgacggcctg ctgatccagc
     9601 cgctgatcgg cagcctgccc aacatcatgg ccccggagtg gttcgacggc ctcaagcgcg
     9661 ccgccgaggg ccgccgcctg atggtgctgg acacgctgcg ccggttccac atcgaggaag
     9721 aaaacgccag cggccccatg gcccaggtca tcggtcgcat ggaggccatc gccgccgata
     9781 ccgggtgctc tatcgtgttc ctgcaccatg ccagcaaggg cgcggccatg atgggcgcag
     9841 gcgaccagca gcaggccagc cggggcagct cggtactggt cgataacatc cgctggcagt
     9901 cctacctgtc gagcatgacc agcgccgagg ccgaggaatg gggtgtggac gacgaccagc
     9961 gccggttctt cgtccgcttc ggtgtgagca aggccaacta tggcgcaccg ttcgctgatc
    10021 ggtggttcag gcggcatgac ggcggggtgc tcaagcccgc cgtgctggag aggcagcgca
    10081 agagcaaggg ggtgccccgt ggtgaagcct aagaacaagc acagcctcag ccacgtccgg
    10141 cacgacccgg cgcactgtct ggcccccggc ctgttccgtg ccctcaagcg gggcgagcgc
    10201 aagcgcagca agctggacgt gacgtatgac tacggcgacg gcaagcggat cgagttcagc
    10261 ggcccggagc cgctgggcgc tgatgatctg cgcatcctgc aagggctggt ggccatggct
    10321 gggcctaatg gcctagtgct tggcccggaa cccaagaccg aaggcggacg gcagctccgg
    10381 ctgttcctgg aacccaagtg ggaggccgtc accgctgatg ccatggtggt caaaggtagc
    10441 tatcgggcgc tggcaaagga aatcggggca gaggtcgata gtggtggggc gctcaagcac
    10501 atacaggact gcatcgagcg cctttggaag gtatccatca tcgcccagaa tggccgcaag
    10561 cggcaggggt ttcggctgct gtcggagtac gccagcgacg aggcggacgg gcgcctgtac
    10621 gtggccctga accccttgat cgcgcaggcc gtcatgggtg gcggccagca tgtgcgcatc
    10681 agcatggacg aggtgcgggc gctggacagc gaaaccgccc gcctgctgca ccagcggctg
    10741 tgtggctgga tcgaccccgg caaaaccggc aaggcttcca tagatacctt gtgcggctat
    10801 gtctggccgt cagaggccag tggttcgacc atgcgcaagc gccgccagcg ggtgcgcgag
    10861 gcgttgccgg agctggtcgc gctgggctgg acggtaaccg agttcgcggc gggcaagtac
    10921 gacatcaccc ggcccaaggc ggcaggctga ccccccccac tctattgtaa acaagacatt
    10981 tttatctttt atattcaatg gcttattttc ctgctaattg gtaataccat ggtaccgctc
    11041 tgccagtgtt acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga
    11101 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
    11161 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
    11221 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
    11281 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta
    11341 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
    11401 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
    11461 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
    11521 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
    11581 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
    11641 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
    11701 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
    11761 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
    11821 aaatcagcat ccatgttgga atttaatcgc ggcctggagc aagacgtttc ccgttgaata
    11881 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
    11941 gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttgt
    12001 tgaataaatc gaacttttgc tgagttgaag gatcagctcg agttaccaat gcttaatcag
    12061 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
    12121 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
    12181 gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
    12241 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
    12301 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
    12361 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
    12421 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
    12481 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
    12541 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
    12601 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
    12661 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
    12721 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
    12781 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
    12841 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
    12901 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
    12961 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
    13021 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
    13081 gcgtatcacg aggcagaatt tcagataaaa aaaatcctta gctttcgcta aggatgattt
    13141 ctg
//
