pPtGE34

LOCUS       pPtGE34             12389 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses Cas9 in Phaeodactylum tricornutum / Encodes elements 
            required for conjugation.
ACCESSION   .
VERSION     .
KEYWORDS    pPtGE34
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12389)
  AUTHORS   Slattery SS, Diamond A, Wang H, Therrien JA, Lant JT, Jazey T, Lee 
            K, Klassen Z, Desgagne-Penix I, Karas BJ, Edgell DR
  TITLE     An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome 
            Editing and Stable Maintenance of Synthetic Pathways in 
            Phaeodactylum tricornutum.
  JOURNAL   ACS Synth Biol. 2018 Jan 8. doi: 10.1021/acssynbio.7b00191.
  PUBMED    29298053
REFERENCE   2  (bases 1 to 12389)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..12389
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(782..1153)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1186..1295
                     /note="incP origin of transfer"
     primer_bind     1572..1588
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1612..1630
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2251..2625
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFVGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLNELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        3627..3812
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             3813..4469
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVL"
     CDS             4933..4998
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5005..5025
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5050..9150
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             9151..9198
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_RNA        9734..9809
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(10156..10172)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10157..10173)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(10201..10219)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10240..10256)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10240..10256)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10253..10275)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10264..10280
                     /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(10288..10318)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10333..10354
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10471..10488)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10642..11230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10722..10741)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11401..12261)
                     /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     12024..12043
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12262..12366)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctgatcgt
      661 cttgccttgc tcgtcggtga tgtacttcac cagctccgcg aagtcgctct tcttgatgga
      721 gcgcatgggg acgtgcttgg caatcacgcg caccccccgg ccgttttagc ggctaaaaaa
      781 gtcatggctc tgccctcggg cggaccacgc ccatcatgac cttgccaagc tcgtcctgct
      841 tctcttcgat cttcgccagc agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg
      901 ggtcgtcggt gagccagagt ttcagcaggc cgcccaggcg gcccaggtcg ccattgatgc
      961 gggccagctc gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga
     1021 cggccagcag gtaggccgac aggctcatgc cggccgccgc cgccttttcc tcaatcgctc
     1081 ttcgttcgtc tggaaggcag tacaccttga taggtgggct gcccttcctg gttggcttgg
     1141 tttcatcagc catccgcttg ccctcatctg ttacgccggc ggtagccggc cagcctcgca
     1201 gagcaggatt cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc
     1261 cgctcgcggg tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag
     1321 gaaagtctac acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata
     1381 ccgaaaaaat cgctataatg accccgaagc agggttatgc agcggaagat gccattcgcc
     1441 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     1501 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     1561 gtcacgacgt tgtaaaacga cggccagtga ccatgattac gccaagctcg aaattaaccc
     1621 tcactaaagg gaacaaaagc tggtacctaa ccctgcgata gaccttttcc aaactcacgc
     1681 agtccaagaa aacaaagggg tgagaagtat acgcaccttt cggtttcggc ataattctta
     1741 aactcttgtg gtcactttct tgtgaagaag ctaggggcac tcgttttccc tcagagcctg
     1801 caaacacaaa attcctgcag tcaattgtcc caacactcgg caagccgtat gcgcaagcaa
     1861 cgatgcgcag aaggccgtgg atggatggcg actcgcgata tggcttcttg ggtcgccagt
     1921 gtggtatgtc cggcgtatgt caatacgcga attcggacga ctggcatctc taggaggagg
     1981 attccttctt ttatgacatg tttattttat atacattgat gctttccgac agtcggaagt
     2041 aataaatgaa tttatttcaa gactacctat actcctttga cttgttcgac taatcttacc
     2101 gcttactaaa atctcgaaat cacgcttgac ctctcgcacg caaatttttg ctgctggacg
     2161 ctacgcactc ggcccaattc ttctcggtcc tcgtcgtcgc aattgtcgtt gcgttgatct
     2221 tgcaccgaag gaatcagaga atagaatacc atggccaagt tgaccagtgc cgttccggtg
     2281 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     2341 cgggacttcg tggaggacga cttcgtcggt gtggtccggg acgacgtgac cctgttcatc
     2401 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     2461 ctgaacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
     2521 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
     2581 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgaagata agaatatctc
     2641 attgtgaaca tctatgattt accaatttta ttctttgttt acagttagac gccagtaatt
     2701 gtgctgtttc tctcaagtct gtgtcaatac aaactacgaa acttggcaat ttttctcttg
     2761 aatatgagca cgagattgaa acgcacaaag gaaattagtt tccatccttt gacaaagttt
     2821 gttgctgttt agagaacaga tgtcaaaatt aacgtgccat ggaattgaac catggtgcgc
     2881 tatccccaaa tcacgtcgtt tgacctcgtc acatttaagg tatatcaagc atattcactt
     2941 atatcttgac atcctccctg cttgatattc ctttgccctt gagccatctt ccccacacgc
     3001 tggagatgac ccctgcccct cctttcttta tccacccgag taaggtgtcg acaagtcact
     3061 ttgcccctga gccatcctcc ccactcagga gatgacccct acccctttct tctttatcca
     3121 cccccacgtg ctataaaaat aattataatt taaatttttt aatataaata tataaattaa
     3181 aaatagaaag taaaaaaaga aattaaagaa aaaatagttt ttgttttccg aagatgtaaa
     3241 agactctagg gggatcgcca acaaatacta ccttttacct tgctcttcct gctctcaggt
     3301 attaatgccg aattgtttca tcttgtctgt gtagaagacc acacacgaaa atcctgtgat
     3361 tttacatttt acttatcgtt aatcgaatgt atatctattt aatctgcttt tcttgtctaa
     3421 taaatatata tgtaaagtac gctttttgtt gaaatttttt aaacctttgt ttattttttt
     3481 ttcttcattc cgtaactctt ctaccttctt tatttacttt ctaaaatcca aatacaaaac
     3541 ataaaaataa ataaacacag agtaaattcc caaattattc catcattaaa agatacgagg
     3601 cgcgtgtaag ttacaggcaa gcgatcctag tacactctat atttttttat gcctcggtaa
     3661 tgattttcat ttttttttcc acctagcgga tgactctttt tttttcttag cgattggcat
     3721 tatcacataa tgaattatac attatataaa gtaatgtgat ttcttcgaag aatatactaa
     3781 aaaatgagca ggcaagataa acgaaggcaa agatgacaga gcagaaagcc ctagtaaagc
     3841 gtattacaaa tgaaaccaag attcagattg cgatctcttt aaagggtggt cccctagcga
     3901 tagagcactc gatcttccca gaaaaagagg cagaagcagt agcagaacag gccacacaat
     3961 cgcaagtgat taacgtccac acaggtatag ggtttctgga ccatatgata catgctctgg
     4021 ccaagcattc cggctggtcg ctaatcgttg agtgcattgg tgacttacac atagacgacc
     4081 atcacaccac tgaagactgc gggattgctc tcggtcaagc ttttaaagag gccctagggg
     4141 ccgtgcgtgg agtaaaaagg tttggatcag gatttgcgcc tttggatgag gcactttcca
     4201 gagcggtggt agatctttcg aacaggccgt acgcagttgt cgaacttggt ttgcaaaggg
     4261 agaaagtagg agatctctct tgcgagatga tcccgcattt tcttgaaagc tttgcagagg
     4321 ctagcagaat taccctccac gttgattgtc tgcgaggcaa gaatgatcat caccgtagtg
     4381 agagtgcgtt caaggctctt gcggttgcca taagagaagc cacctcgccc aatggtacca
     4441 acgatgttcc ctccaccaaa ggtgttcttt atgcaatctc gcctattcat ggtgtataaa
     4501 agttcaacat ccaaagctag aacttttgga aagagaaaga atatccgaat agggcacggc
     4561 gtgccgtatt gttggagtgg actagcagaa agtgaggaag gcacaggatg agttttctcg
     4621 agacacatag cttcagcgtc gtgtaggcta ggcagaggtg agttttctcg agacatacct
     4681 tcagcgtcgt cttcactgtc acagtcaact gacagtaatc gttgatccgg agagattcaa
     4741 aattcaatct gtttggacct ggataagaca caagagcgac atcctgacat gaacgccgta
     4801 aacagcaaat cctggttgaa cacgtatcct tttgggggcc tccgctacga cgctcgctcc
     4861 agctggggct tccttactat acacagcgcg catatttcac ggttgccaga agtcaagatg
     4921 gcctctagaa tggactataa ggaccacgac ggagattaca aggatcatga tattgattac
     4981 aaagacgatg atgataaaat ggccccaaag aagaagcgga aagtcggtat acacggagtc
     5041 ccagcagccg acaagaagta ctcgattgga ttggacatcg gcaccaactc tgttggatgg
     5101 gccgtcatca cggacgagta caaggtgccc agcaagaaat tcaaagtact gggaaacact
     5161 gaccgccatt cgatcaagaa gaatttgatt ggagctttgc tgtttgacag cggcgaaaca
     5221 gcggaagcca cgcggcttaa acgaaccgcg agacgaagat atacgcgacg gaagaaccgt
     5281 atctgctatc tacaagagat tttctcgaat gagatggcaa aagtcgatga ctctttcttt
     5341 caccgattgg aagaatcctt cctcgttgaa gaggataaga aacatgagcg gcacccgata
     5401 tttggaaaca ttgtggacga ggtggcttac catgagaaat acccgactat ttaccacctg
     5461 cgaaagaaat tggtggactc gacggacaaa gcggatcttc gcctaatcta tttggcgcta
     5521 gcacatatga ttaagtttcg cggccacttc ttgatcgaag gcgacttgaa tccggacaac
     5581 agtgatgttg acaaactgtt tattcaactt gtacaaactt acaaccagct ctttgaagaa
     5641 aatcccatta acgcttcggg agtggacgcg aaagccatcc tttctgcccg actcagcaag
     5701 agccgacgcc tagaaaattt gattgcgcaa cttcccggcg agaaaaagaa tggacttttt
     5761 ggaaatctta ttgctttgtc gctgggcctt acgccgaact ttaagagcaa cttcgacctt
     5821 gcggaagatg ccaaattgca actgagtaaa gacacatatg acgacgacct cgacaatctt
     5881 ttggcacaaa taggagatca gtacgccgac ctctttttgg cggctaagaa cttgtccgac
     5941 gccattctgt tgtcggacat ccttcgagtt aatacggaga ttactaaggc gcccttgagc
     6001 gcctctatga tcaaaagata cgacgagcat caccaagacc tgaccctcct taaagctctc
     6061 gtccggcaac agctgcctga gaagtacaaa gagattttct ttgatcaaag caaaaacgga
     6121 tacgcgggct acattgacgg gggagcctcg caagaagagt tctacaagtt tattaaaccg
     6181 atcctggaaa agatggacgg cacggaggaa ttactcgtga aacttaaccg agaggacctt
     6241 ttgcgtaagc aacggacttt cgacaacgga tctattccgc accaaatcca tctcggagaa
     6301 ttgcacgcaa ttctgcgccg gcaagaagat ttttatccat ttctcaagga caaccgggaa
     6361 aagattgaga aaatcttaac cttccgcata ccctactacg tcggcccttt ggccagggga
     6421 aattcgagat ttgcctggat gacgcgaaag agtgaggaaa ctattactcc ctggaatttc
     6481 gaagaagtgg tagacaaagg agcttccgcg caatccttta tcgagcgcat gaccaacttc
     6541 gataagaacc tcccgaatga gaaagtccta cccaagcatt cgttgctcta cgaatacttt
     6601 acggtgtata atgagctgac taaagtcaaa tacgttacag agggaatgcg aaaaccggct
     6661 ttcctctcgg gagagcagaa aaaggccata gtcgatcttc tctttaaaac gaaccgtaaa
     6721 gttaccgtga aacaactaaa agaggactac ttcaagaaaa tcgaatgctt tgactccgtt
     6781 gaaatttccg gcgttgaaga tcggttcaat gcctccttgg gaacatacca cgatctcctg
     6841 aaaattatca aagacaagga ctttcttgac aatgaggaaa acgaggacat tcttgaagat
     6901 attgtcttga cgctaacact ttttgaggat cgagagatga ttgaggaacg ccttaaaacc
     6961 tatgcgcatc tgttcgacga caaagttatg aagcaattga agcgccggag atatacggga
     7021 tggggaaggt tgagtcggaa actgataaat ggcatacgcg ataagcagtc cggcaaaaca
     7081 attctcgatt tcttgaagtc cgacggattt gcgaatcgaa acttcatgca actgatccat
     7141 gacgattcgt tgacctttaa agaggacatt caaaaagccc aagtatccgg acaaggagat
     7201 tctttgcacg agcatattgc gaatcttgct ggaagtccgg ctattaaaaa gggcattctc
     7261 cagacagtaa aggtcgtgga tgaactcgtt aaagttatgg gccgacacaa acccgagaac
     7321 atagtcatcg aaatggcgag agagaaccag accacccaaa agggacagaa aaattcacgc
     7381 gagcgaatga agcggattga agaggggatc aaagagcttg gcagccaaat tctgaaagaa
     7441 cacccggttg aaaacaccca attgcagaac gagaagctct acctgtacta tcttcaaaat
     7501 gggcgtgata tgtacgtcga tcaagaattg gacatcaacc ggctatccga ttacgatgtg
     7561 gaccatattg ttcctcagtc gtttctcaaa gacgactcca tagacaataa ggttctaacg
     7621 agaagcgaca agaaccgcgg caaatcagac aacgtcccct ccgaggaagt agttaagaaa
     7681 atgaagaatt actggcgcca attgctgaac gctaagctca ttacccaaag aaagtttgac
     7741 aatttgacga aagccgagcg aggcgggctc tcggaactgg ataaggccgg cttcattaaa
     7801 agacaattgg tggaaacgcg acaaatcaca aagcacgtcg cacagattct cgactcccgc
     7861 atgaacacta agtatgacga gaatgacaaa ctaatccggg aagtcaaagt gattaccttg
     7921 aagtccaagc tggtttccga ttttcgtaaa gattttcagt tttacaaagt tcgcgagatc
     7981 aacaactacc atcacgcgca cgacgcttac ttgaacgctg tcgtgggaac ggccttgatt
     8041 aaaaaatacc ctaagctgga aagcgagttc gtttacggcg attacaaggt gtatgacgta
     8101 cgcaaaatga tcgcgaagtc agagcaggaa attggcaaag ctactgcgaa gtacttcttt
     8161 tatagcaaca taatgaattt tttcaaaacg gagattaccc tcgctaacgg cgagattcgc
     8221 aagcgccctc ttatcgagac aaacggcgaa acgggggaga ttgtctggga taaaggccgc
     8281 gattttgcga ctgtgcggaa agtcttgtcg atgccgcaag tgaatatagt caaaaagacg
     8341 gaggtgcaaa caggcggctt ttcgaaagaa tctatccttc ccaaaaggaa ctctgataag
     8401 ttgattgcgc gaaaaaagga ttgggaccct aagaagtatg gcggctttga cagcccgacc
     8461 gtagcatatt ctgtccttgt ggtggccaaa gtcgaaaagg gcaaatccaa gaaactgaag
     8521 agtgtgaaag aattgcttgg gattacgatc atggaaagat catcttttga gaagaatccc
     8581 atcgactttc tcgaagctaa aggctacaaa gaagtgaaaa aggacctgat tatcaagttg
     8641 cctaagtact ccttgtttga gctggaaaat ggccgtaagc gaatgctcgc ttctgccggc
     8701 gaactccaga agggaaacga attggcgctg ccctccaaat atgttaactt tctttacctt
     8761 gcctcgcact atgagaagct caagggctcc cccgaggata atgagcaaaa acaactgttt
     8821 gtagaacagc ataagcacta cttggacgag atcattgagc aaattagcga gttctccaag
     8881 cgagtcatcc ttgccgacgc taatctggac aaagtcctgt ccgcctacaa taagcaccgc
     8941 gataagccaa tccgagagca agcggagaat attatccact tgtttacgct caccaatctt
     9001 ggagcccctg ccgcctttaa gtactttgac accaccatcg accgcaagag gtacaccagc
     9061 accaaagaag tcttggacgc caccctcatt caccaatcaa tcaccggctt gtacgagaca
     9121 cgcatcgacc tgtctcaact cggaggcgac aaaaggccgg cggccacgaa aaaggccggc
     9181 caggcaaaaa agaaaaagta atctggatcc ctacctcgac tttggctggg acactttcag
     9241 tgaggacaag aagctccaga agcgtgctat cgaactcaac cagggacgtg cggcacaaat
     9301 gggcatcctt gctctcatgg tgcacgaaca gttgggagtc tctatccttc cttaaaaatt
     9361 taattttcat tagttgcagt cactccgctt tggtttcaca gtcaggaata acactagctc
     9421 gtcttcactg caggttggct cggaagttgg tgttgacggt gagctggaaa ttggttgtcg
     9481 gtcactgcta gcgagaagaa aacggaggac agaaggaagt gaaactcggt tcgttctcga
     9541 cagcctcact gtcaatatgc tcattttcaa tccttagcgc ttttaatgtc gaattgacgg
     9601 taaattgaat aggatctata atatctacaa ggtactttga cacgccaagt attcattgtt
     9661 agtcaacaat attttagagc tttataaggt caaaaaacac cttcaaagtc gaggagaccg
     9721 agagagggtc tcagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     9781 aacttgaaaa agtggcaccg agtcggtgct tttttttaga accgctcacc catgctatcg
     9841 tatgtcattt acattgacaa actgctatga atgcgtttgc gataacttca gctcgctgaa
     9901 gccctagaca tttcgtttga ctaaattgtt ttccggttgc tttactaccg accggtctgc
     9961 gggtagcatg tcttggcttt tgcgagttcg agagaattta tggacaagtg cgtttacagt
    10021 ttggaacacg agtgtgagtg agcggaattg gctatctata acattcttat ttcaggaggc
    10081 tggtgattag cagaattttc aaagatctcg aatctaaaat cgccatgggc tcgacgaact
    10141 tttctgcaag cttatcgata ccgtcgacct cgaggggggg cccggtacca gcttttgttc
    10201 cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
    10261 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
    10321 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
    10381 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
    10441 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
    10501 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
    10561 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
    10621 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
    10681 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
    10741 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
    10801 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
    10861 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
    10921 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
    10981 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
    11041 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
    11101 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
    11161 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
    11221 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
    11281 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
    11341 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
    11401 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
    11461 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
    11521 cagtgctgca atgataccgc gagagccacg ctcaccggct ccagatttat cagcaataaa
    11581 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
    11641 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
    11701 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
    11761 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
    11821 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
    11881 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
    11941 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
    12001 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
    12061 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
    12121 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
    12181 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
    12241 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
    12301 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
    12361 tccgcgcaca tttccccgaa aagtgccac
//