LOCUS       sequence-412308-       13492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RP4-based mobilizable plasmid for the IPTG inducible expression of 
            inserts that can be introduced using BsaI-based Golden Gate cloning.
            Vector has two origins of replication..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13492)
  AUTHORS   Bobonis J, Yang ALJ, Voogdt CGP, Typas A
  TITLE     TAC-TIC, a high-throughput genetics method to identify triggers or 
            blockers of bacterial toxin-antitoxin systems.
  JOURNAL   Nat Protoc. 2024 May 9. doi: 10.1038/s41596-024-00988-y.
  PUBMED    38724726
REFERENCE   2  (bases 1 to 13492)
  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: "Nat Protoc.
            2024 May 9. doi: 10.1038/s41596-024-00988-y."
COMMENT     Sequence Label: pJB158
FEATURES             Location/Qualifiers
     source          1..13492
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    58..531
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
     CDS             complement(604..1575)
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     CDS             complement(1575..2750)
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     misc_feature    3076..3326
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(3329..4084)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      4175..4394
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     CDS             complement(5665..5970)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(5767..5786)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     oriT            6485..6594
                     /note="incP origin of transfer"
     promoter        6765..6842
                     /gene="lacI"
                     /label=lacI promoter
     CDS             6843..7925
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6862..6881)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7962..7976,7977..7982,7983..7999,8000..8005)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    7982..7998
                     /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)."
     protein_bind    8012..8031
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     RBS             8048..8059
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             8232..8243
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             8250..8963
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      8975..9046
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      9062..9089
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      9172..9266
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(9336..9358)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        join(9603..9608,9609..9625,9626..9631)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             9801..10334
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     CDS             complement(11178..11804)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     11709..11728
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        11820..11875
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    11826..11844
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    11856..11874
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             11883..13088
                     /codon_start=1
                     /gene="tetA from transposon Tn10"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MNSSTKIALVITLLDAMGIGLIMPVLPTLLREFIASEDIANHFGV
                     LLALYALMQVIFAPWLGKMSDRFGRRPVLLLSLIGASLDYLLLAFSSALWMLYLGRLLS
                     GITGATGAVAASVIADTTSASQRVKWFGWLGASFGLGLIAGPIIGGFAGEISPHSPFFI
                     AALLNIVTFLVVMFWFRETKNTRDNTDTEVGVETQSNSVYITLFKTMPILLIIYFSAQL
                     IGQIPATVWVLFTENRFGWNSMMVGFSLAGLGLLHSVFQAFVAGRIATKWGEKTAVLLG
                     FIADSSAFAFLAFISEGWLVFPVLILLAGGGIALPALQGVMSIQTKSHQQGALQGLLVS
                     LTNATGVIGPLLFAVIYNHSLPIWDGWIWIIGLAFYCIIILLSMTFMLTPQAQGSKQET
                     SA"
ORIGIN
        1 aatacttgcc cttgacaggc attgatggaa tcgtagtctc acgctgatag tctgatcgac
       61 aatacaagtg ggaccgtggt cccagaccga taatcagacc gacaacacga gtgggatcgt
      121 ggtcccagac taataatcag accgacgata cgagtgggac cgtggtccca gactaataat
      181 cagaccgacg atacgagtgg gaccgtggtt ccagactaat aatcagaccg acgatacgag
      241 tgggaccgtg gtcccagact aataatcaga ccgacgatac gagtgggacc atggtcccag
      301 actaataatc agaccgacga tacgagtggg accgtggtcc cagtctgatt atcagaccga
      361 cgatacgagt gggaccgtgg tcccagacta ataatcagac cgacgatacg agtgggaccg
      421 tggtcccaga ctaataatca gaccgacgat acgagtggga ccgtggtccc agtctgatta
      481 tcagaccgac gatacaagtg gaacagtggg cccagagaga atattcaggc cagttatgct
      541 ttctggcctg taacaaagga cattaagtaa agacagataa acgtagacta aaacgtggtc
      601 gcatcagggt gctggctttt caagttcctt aagaatggcc tcaattttct ctatacactc
      661 agttggaaca cgtgacctgt ccaggttaag caccatttta tcgcccttat acaatactgt
      721 cgctccagga gcaaactgat gtcgtgagct taaactagtt cttgatgcag atgacgtttt
      781 aagcacagaa gttaaaagag tgataacttc ttcagcttca aatatcaccc cagctttttt
      841 ctgctcatga aggttagatg cctgctgctt aagtaattcc tctttatctg taaaggcttt
      901 ttgaagtgca tcacctgacc gggcagatag ttcaccgggg tgagaaaaaa gagcaacaac
      961 tgatttaggc aatttggcgg tgttgataca gcgggtaata atcttacgtg aaatattttc
     1021 cgcatcagcc agcgcagaaa tatttccagc aaattcattc tgcaatcggc ttgcataacg
     1081 ctgaccacgt tcataagcac ttgttgggcg ataatcgtta cccaatctgg ataatgcagc
     1141 catctgctca tcatccagct cgccaaccag aacacgataa tcactttcgg taagtgcagc
     1201 agctttacga cggcgactcc catcggcaat ttctatgaca ccagatactc ttcgaccgaa
     1261 cgccggtgtc tgttgaccag tcagtagaaa agaagggatg agatcatcca gtgcgtcctc
     1321 agtaagcagc tcctggtcac gttcattacc tgaccatacc cgagaggtct tctcaacact
     1381 atcaccccgg agcacttcaa gagtaaactt cacatcccga ccacatacag gcaaagtaat
     1441 ggcattaccg cgagccatta ctcctacgcg cgcaattaac gaatccacca tcggggcagc
     1501 tggtgtcgat aacgaagtat cttcaaccgg ttgagtattg agcgtatgtt ttggaataac
     1561 aggcgcacgc ttcattatct aatctcccag cgtggtttaa tcagacgatc gaaaatttca
     1621 ttgcagacag gttcccaaat agaaagagca tttctccagg caccagttga agagcgttga
     1681 tcaatggcct gttcaaaaac agttctcatc cggatctgac ctttaccaac ttcatccgtt
     1741 tcacgtacaa cattttttag aaccatgctt ccccaggcat cccgaatttg ctcctccatc
     1801 cacggggact gagagccatt actattgctg tatttggtaa gcaaaatacg tacatcaggc
     1861 tcgaaccctt taagatcaac gttcttgagc agatcacgaa gcatatcgaa aaactgcagt
     1921 gcggaggtgt agtcaaacaa ctcagcaggc gtgggaacaa tcagcacatc agcagcacat
     1981 acgacattaa tcgtgccgat acccaggtta ggcgcgctgt caataactat gacatcatag
     2041 tcatgagcaa cagtttcaat ggccagtcgg agcatcaggt gtggatcggt gggcagttta
     2101 ccttcatcaa atttgcccat taactcagtt tcaatacggt gcagagccag acaggaagga
     2161 ataatgtcaa gccccggcca gcaagtgggc tttattgcat aagtgacatc gtccttttcc
     2221 ccaagataga aaggcaggag agtgtcttct gcatgaatat gaagatctgg tacccatccg
     2281 tgatacattg aggctgttcc ctgggggtcg ttaccttcca cgagcaaaac acgtagcccc
     2341 ttcagagcca gatcctgagc aagatgaaca gaaactgagg ttttgtaaac gccaccttta
     2401 tgggcagcaa ccccgatcac cggtggaaat acgtcttcag cacgtcgcaa tcgcgtacca
     2461 aacacatcac gcatatgatt aatttgttca attgtataac caacacgttg ctcaacccgt
     2521 cctcgaattt ccatatccgg gtgcggtagt cgccctgctt tctcggcatc tctgatagcc
     2581 tgagaagaaa ccccaactaa atccgctgct tcacctattc tccagcgccg ggttattttc
     2641 ctcgcttccg ggctgtcatc attaaactgt gcaatggcga tagccttcgt catttcatga
     2701 ccagcgttta tgcactggtt aagtgtttcc atgagtttca ttctgaacat cctttaatca
     2761 ttgctttgcg tttttttatt aaatcttgca atttactgca aagcaacaac aaaatcgcaa
     2821 agtcatcaaa aaaccgcaaa gttgtttaaa ataagagcaa cactacaaaa ggagataaga
     2881 agagcacata cctcagtcac ttattatcac tagcgctcgc cgcagccgtg taaccgagca
     2941 tagcgagcga actggcgagg aagcaaagaa gaactgttct gtcagatagc tcttacgctc
     3001 agcgcaagaa gaaatatcca ccgtgggaaa aactccaggt agaggtacac acgcggatag
     3061 ccaattcaga gtaataaact gtgataatca accctcatca atgatgacga actaaccccc
     3121 gatatcaggt cacatgacga agggaaagag aaggaaatca actgtgacaa actgccctca
     3181 aatttggctt ccttaaaaat tacagttcaa aaagtatgag aaaatccatg caggctgaag
     3241 gaaacagcaa aactgtgaca aattaccctc agtaggtcag aacaaatgtg acgaaccacc
     3301 ctcaaatctg tgacagataa ccctcagact atcctgtcgt catggaagtg atatcgcgga
     3361 aggaaaatac gatatgagtc gtctggcggc ctttcttttt ctcaatgtat gagaggcgca
     3421 ttggagttct gctgttgatc tcattaacac agacctgcag gaagcggcgg cggaagtcag
     3481 gcatacgctg gtaactttga ggcagctggt aacgctctat gatccagtcg attttcagag
     3541 agacgatgcc tgagccatcc ggcttacgat actgacacag ggattcgtat aaacgcatgg
     3601 catacggatt ggtgatttct tttgtttcac taagccgaaa ctgcgtaaac cggttctgta
     3661 acccgataaa gaagggaatg agatatgggt tgatatgtac actgtaaagc cctctggatg
     3721 gactgtgcgc acgtttgata aaccaaggaa aagattcata gcctttttca tcgccggcat
     3781 cctcttcagg gcgataaaaa accacttcct tccccgcgaa actcttcaat gcctgccgta
     3841 tatccttact ggcttccgca gaggtcaatc cgaatatttc agcatattta gcaacatgga
     3901 tctcgcagat accgtcatgt tcctgtaggg tgccatcaga ttttctgatc tggtcaacga
     3961 acagatacag catacgtttt tgatcccggg agagactata tgccgcctca gtgaggtcgt
     4021 ttgactggac gattcgcggg ctatttttac gtttcttgtg attgataacc gctgtttccg
     4081 ccatgacaga tccatgtgaa gtgtgacaag tttttagatt gtcacactaa ataaaaaaga
     4141 gtcaataagc agggataact ttgtgaaaaa acagcttctt ctgagggcaa tttgtcacag
     4201 ggttaagggc aatttgtcac agacaggact gtcatttgag ggtgatttgt cacactgaaa
     4261 gggcaatttg tcacaacacc ttctctagaa ccagcatgga taaaggccta caaggcgctc
     4321 taaaaaagaa gatctaaaaa ctataaaaaa aataattata aaaatatccc cgtggataag
     4381 tggataaccc caagggaagt tttttcaggc atcgtgtgta agcagaatat ataagtgctg
     4441 ttccctggtg cttcctcgct cactcgaggg cttcgccctg tcgctcgact gcggcgagca
     4501 ctactggctg taaaaggaca gaccacatca tggttctgtg ttcattaggt tgttctgtcc
     4561 attgctgaca taatccgctc cacttcaacg taacaccgca cgaagatttc tattgttcct
     4621 gaaggcatat tcaaatcgtt ttcgttaccg cttgcaggca tcatgacaga acactacttc
     4681 ctataaacgc tacacaggct cctgagatta ataatgcgga tctctacgat aatgggagat
     4741 tttcccgact gtttcgttcg cttctcagtg gataacagcc agcttctctg tttaacagac
     4801 aaaaacagca tatccactca gttccacatt tccatataaa ggccaaggca tttattctca
     4861 ggataattgt ttcagcatcg caaccgcatc agactccggc atcgcaaact gcacccggtg
     4921 ccgggcagcc acatccagcg caaaaacctt cgtgtagact tccgttgaac tgatggactt
     4981 atgtcccatc aggctttgca gaactttcag cggtataccg gcatacagca tgtgcatcgc
     5041 ataggaatgg cggaacgtat gtggtgtgac cggaacagag aacgtcacac cgtcagcagc
     5101 agcggcggca accgcctccc caatccaggt cctgaccgtt ctgtccgtca cttcccagat
     5161 ccgcgctttc tctgtccttc ctgtgcgacg gttacgccgc tccatgggta ttttcagtgt
     5221 tgccaccatc gtctgcagct ggctgacgta ccaggagtca gagagcggaa ccagccggtg
     5281 agtctgctga ccggcgggca ttctccccgc cgtcctggca gctttttcgg tccgttgttt
     5341 cagggtcgca agctgcacaa acggatacgg aggcgcaagc gaaaaatccc cccgcgtcag
     5401 cgccagtgct tcattaatgc gtgctccggt gttccacagt gtggccagca gcatcttgcg
     5461 gtgcagatcc gggacgtaat ggagcagggc actcacttcc ggagccagca gatattttgg
     5521 cagttcatca tggaccatcg acatctggcg aagtgccaga gctgccggat aatcaatggc
     5581 aacaggcagc gatgcaggct gcccggcaga atacactgcc ggtaccatga ctgcagactg
     5641 gctgtgtata acggagcctg acatttatat tccccagaac atcaggttaa tggcgttttt
     5701 gatgtcattt tcgcggtggc tgagatcagc cacttcttcc ccgataacgg acaccggcac
     5761 actggccata tcggtggtca tcatgcgcca gctttcatcc ccgatatgca ccaccgggta
     5821 aagttcacgg gagactttat ctgacagcag acgtgcactg gccaggggga tcaccatccg
     5881 tcgcccgggc gtgtcaataa tatcactctg tacatccaca aacagacgat aacggctctc
     5941 tcttttatag gtgtaaacct taaactgcat ttcaccagtc cctgttctcg tcagcaaaag
     6001 agccgttcat ttcaataaac cgggcgacct cagccatccc ttcctgattt tccgctttcc
     6061 agcgttcggc acgcagacga cgggcttcat tctgcatggt tgtgcttacc agaccggaga
     6121 tattgacatc atatgccttg agcaactgat agctgtcgct gtcaactgtc actgtaatac
     6181 gctgcttcat agcacacctc tttttgacat acttcgggta tacatatcag tatatattct
     6241 tataccgcaa aaatcagcgc gcaaatacgc atactgttat ctggctttta gtaagcctta
     6301 tgtattttac ctttcgttat gttaaccaat aaaaattaaa atctgactta taaaaacaaa
     6361 gcgtaattac cgcattcccg tttcgtatgg atccggatca attccgtgat aggtgggctg
     6421 cccttcctgg ttggcttggt ttcatcagcc atccgcttgc cctcatctgt tacgccggcg
     6481 gtagccggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg cgaataaggg
     6541 acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct gcccggctga
     6601 cgccgttgga tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt
     6661 gcgaaaaagg atggatatac cgaaaaaatc gctataatga ccccgaagca gggttatgca
     6721 gcggaaaacg gaattgatcc ggatcctcac accgcataaa ttccgacacc atcgaatggc
     6781 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     6841 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     6901 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     6961 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     7021 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     7081 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     7141 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     7201 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     7261 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     7321 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     7381 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     7441 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     7501 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     7561 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     7621 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     7681 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     7741 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     7801 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     7861 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     7921 agtgataacg gaccgcggcc tttcgtcttc acctcgagaa atcataaaaa atttatttgc
     7981 tttgtgagcg gataacaatt ataatgtgtg aaattgtgag cgctcacaat ttcacacaga
     8041 attcattaaa gaggagaaac ctaggaggtt cagcatcgag accgaaagtg aaacgtgatt
     8101 tcatgcgtca ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg caattcacat
     8161 ttaatttatg aatgttttct taacatcgcg gcaactcaag aaacggcagg ttcggatctt
     8221 agctactaga gaaagaggag aaatactaga tgcgtaaagg cgaagagctg ttcactggtg
     8281 tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt tccgtgcgtg
     8341 gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc tgtactactg
     8401 gtaaactgcc ggttccttgg ccgactctgg taacgacgct gacttatggt gttcagtgct
     8461 ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc atgccggaag
     8521 gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa acgcgtgcgg
     8581 aagtgaaatt tgaaggcgat accctggtaa accgcattga gctgaaaggc attgacttta
     8641 aagaggacgg caatatcctg ggccataagc tggaatacaa ttttaacagc cacaatgttt
     8701 acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt cgccacaacg
     8761 tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca atcggtgatg
     8821 gtcctgttct gctgccagac aatcactatc tgagcacgca aagcgttctg tctaaagatc
     8881 cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg ggcatcacgc
     8941 atggtatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa
     9001 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     9061 tggctcacct tcgggtgggc ctttctgcgt ttataggtct cgtaagatag ctggccggcc
     9121 tatgcggccg ctaagggtcg acctgcagcc aagcttaatt agctgagctt ggactcctgt
     9181 tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc
     9241 gccgggcgtt ttttattggt gagaatccaa gctaggggaa ttcatcgtga ctgactgacg
     9301 atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     9361 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     9421 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     9481 tataattctt gaagacgaaa gggcctcgtg atacgctcgc acgggcccat ctggcagttc
     9541 cctactctcg catggggagt ccccacacta ccatcggggg ccacaggcgc ccacgaaccc
     9601 agttgacata agcctgttcg gttcgtaaac tgtaatgcaa gtagcgtatg cgctcacgca
     9661 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
     9721 cttgttatga ctgttttttt gtacagtcta tgcctcgggc atccaagcag caagcgcgtt
     9781 acgccgtggg tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc
     9841 gccctaaaac aaagttaggt ggctcaagta tgggcatcat tcgcacatgt aggctcggcc
     9901 ctgaccaagt caaatccatg cgggctgctc ttgatctttt cggtcgtgag ttcggagacg
     9961 tagccaccta ctcccaacat cagccggact ccgattacct cgggaacttg ctccgtagta
    10021 agacattcat cgcgcttgct gccttcgacc aagaagcggt tgttggcgct ctcgcggctt
    10081 acgttctgcc caagtttgag cagccgcgta gtgagatcta tatctatgat ctcgcagtct
    10141 ccggagagca ccggaggcag ggcattgcca ccgcgctcat caatctcctc aagcatgagg
    10201 ccaacgcgct tggtgcttat gtgatctacg tgcaagcaga ttacggtgac gatcccgcag
    10261 tggctctcta tacaaagttg ggcatacggg aagaagtgat gcactttgat atcgacccaa
    10321 gtaccgccac ctaacaattc gttcaaggcg gccgccacct ctggtgactt tatccgtaaa
    10381 taatttaacc cactccacaa aaaggctcaa caggttggtg gttctcacca ccaaaagcac
    10441 cacaccccac gcaaaaacaa gtttttgctg atttttcttt ataaatagag tgttatgaaa
    10501 aattagtttc tcttactctc tttatgatat ttaaaaaagc ggtgtcggcg cggctacaac
    10561 aacgcgccga caccgttttg taggggtggt actgactatt tttataaaaa acattatttt
    10621 atattagggg tgctgctagt ggcgcggtgt gtttttttat aggataccgc taggggcgct
    10681 gcgcggccgc aagaggctaa ttcccatgtc agccgttaag tgttcctgtg tcactcaaaa
    10741 ttgctttgag aggctctaag ggcttctcag tgcgttacat ccctggcttg ttgtccacaa
    10801 ccgttaaacc ttaaaagctt taaaagcctt atatattctt ttttttctta taaaacttaa
    10861 aaccttagag gctatttaag ttgctgattt atattaattt tattgttcaa acatgagagc
    10921 ttagtacgtg aaacatgaga gcttagtacg tgaaacatga gagcttagta cgttagccat
    10981 gagagcttag tacgttagcc atgagggttt agttcgttaa acatgagagc ttagtacgtt
    11041 aaacatgaga gcttagtacg tgaaacatga gagcttagta cgtactatca acaggttgaa
    11101 ctgctgatct tcagatcctc tacgccggac gcttggttac cgtgaagtta ccatcacgga
    11161 aaaaggttat gctgctttta agacccactt tcacatttaa gttgtttttc taatccgcat
    11221 atgatcaatt caaggccgaa taagaaggct ggctctgcac cttggtgatc aaataattcg
    11281 atagcttgtc gtaataatgg cggcatacta tcagtagtag gtgtttccct ttcttcttta
    11341 gcgacttgat gctcttgatc ttccaatacg caacctaaag taaaatgccc cacagcgctg
    11401 agtgcatata atgcattctc tagtgaaaaa ccttgttggc ataaaaaggc taattgattt
    11461 tcgagagttt catactgttt ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg
    11521 cgatgactta gtaaagcaca tctaaaactt ttagcgttat tacgtaaaaa atcttgccag
    11581 ctttcccctt ctaaagggca aaagtgagta tggtgcctat ctaacatctc aatggctaag
    11641 gcgtcgagca aagcccgctt attttttaca tgccaataca atgtaggctg ctctacacct
    11701 agcttctggg cgagtttacg ggttgttaaa ccttcgattc cgacctcatt aagcagctct
    11761 aatgcgctgt taatcacttt acttttatct aatctagaca tcattaattc ctaatttttg
    11821 ttgacactct atcattgata gagttatttt accactccct atcagtgata gagaaaagtg
    11881 aaatgaatag ttcgacaaag atcgcattgg taattacgtt actcgatgcc atggggattg
    11941 gccttatcat gccagtcttg ccaacgttat tacgtgaatt tattgcttcg gaagatatcg
    12001 ctaaccactt tggcgtattg cttgcacttt atgcgttaat gcaggttatc tttgctcctt
    12061 ggcttggaaa aatgtctgac cgatttggtc ggcgcccagt gctgttgttg tcattaatag
    12121 gcgcatcgct ggattactta ttgctggctt tttcaagtgc gctttggatg ctgtatttag
    12181 gccgtttgct ttcagggatc acaggagcta ctggggctgt cgcggcatcg gtcattgccg
    12241 ataccacctc agcttctcaa cgcgtgaagt ggttcggttg gttaggggca agttttgggc
    12301 ttggtttaat agcggggcct attattggtg gttttgcagg agagatttca ccgcatagtc
    12361 ccttttttat cgctgcgttg ctaaatattg tcactttcct tgtggttatg ttttggttcc
    12421 gtgaaaccaa aaatacacgt gataatacag ataccgaagt aggggttgag acgcaatcga
    12481 attcggtata catcacttta tttaaaacga tgcccatttt gttgattatt tatttttcag
    12541 cgcaattgat aggccaaatt cccgcaacgg tgtgggtgct atttaccgaa aatcgttttg
    12601 gatggaatag catgatggtt ggcttttcat tagcgggtct tggtctttta cactcagtat
    12661 tccaagcctt tgtggcagga agaatagcca ctaaatgggg cgaaaaaacg gcagtactgc
    12721 tcggatttat tgcagatagt agtgcatttg cctttttagc gtttatatct gaaggttggt
    12781 tagttttccc tgttttaatt ttattggctg gtggtgggat cgctttacct gcattacagg
    12841 gagtgatgtc tatccaaaca aagagtcatc agcaaggtgc tttacaggga ttattggtga
    12901 gccttaccaa tgcaaccggt gttattggcc cattactgtt tgctgttatt tataatcatt
    12961 cactaccaat ttgggatggc tggatttgga ttattggttt agcgttttac tgtattatta
    13021 tcctgctatc gatgaccttc atgttaaccc ctcaagctca ggggagtaaa caggagacaa
    13081 gtgcttagtt atttcgtcac caaatgatgt tattccgcga aatattacgc ctgaataagt
    13141 gataataagc ggatgaatgg cagaaattcg aattctcctc tcacatgcgg cctccatcag
    13201 gccgtgccgc acctgtaacc gggcgaccgc atacgaatgc aggtgcggcc ccgaagccgc
    13261 agcgcgtgga ggagaaacga cgaaacgtga ccgggtgagg aggccgtcga cagcgacaca
    13321 cttgcatcgg atgcagcccg gttaacgtgc cggcacggcc tgggtaacca ggtattttgt
    13381 ccacataacc gtgcgcaaaa tgttgtggat aagcaggaca cagcagcaat ccacagcagg
    13441 catacaaccg cacaccgagg ttactccgtt ctacaggtta cgacgacatg tc
//
