LOCUS       sequence-202027-       12397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9-HA and sgRNA. Hygromycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12397)
  AUTHORS   Poliner E, Takeuchi T, Du ZY, Benning C, Farre EM
  TITLE     Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR 
            System in the Oleaginous Microalga, Nannochloropsis oceanica 
            CCMP1779.
  JOURNAL   ACS Synth Biol. 2018 Apr 20;7(4):962-968. doi: 
            10.1021/acssynbio.7b00362. Epub 2018 Mar 14.
  PUBMED    29518315
REFERENCE   2  (bases 1 to 12397)
  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"; doi: 
            "10.1021/acssynbio.7b00362"; journalName: "ACS Synth Biol"; date: 
            "2018-04-20- 20"; volume: "7"; issue: "4"; pages: "962-968"
COMMENT     Sequence Label: pNOC-CRISPR-HA
FEATURES             Location/Qualifiers
     source          1..12397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           complement(1430..1497)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_RNA        complement(1502..1577)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2568..2579
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2800..2820
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2821..6921
                     /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             2821..6921
                     /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             2821..6921
                     /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             6958..6984
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6988..7008
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7689..7698
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8528..9553
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             8528..9553
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(9789..9806)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9960..10548)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10040..10059)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10719..11510,11511..11579))
                     /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     11342..11361
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11580..11684)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11762..12217)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11899..11920)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12111..12130
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12343..12365
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12357..12374
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12358..12374
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(12381..12397,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(12381..12397,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt
      121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc
      181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt
      241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg
      301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga
      361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtgca
      421 aagctgacgc ccttttcgtc gtcgttgctg gtgttaaaat tgcgcgtttc ttttttttcc
      481 gtctttgtcg tcttttgact tttttatctt cttttctgtc ttcttttatt cttctcttct
      541 tcttcatcac tccctcttca tttttcttct tctttttcct cgtctattgt cctcgggcat
      601 taccagttgt gctgctcacg tgcgcgcaag cggtgaaagg gtgagaggat acacggacaa
      661 caatcattga aaattacgct taaagccggt gcctggaaaa gtccacgcgc agatggcctt
      721 tgcttgcctt gctgctgctg cagctgctgc tgctgtcgcc tgtgatggta atggttgttg
      781 ttgtggtcct gcttgcttgc tagtttgcat tgcgtttgcg gttgccttat gaccgactcc
      841 gtgtgattga tccttaaaca tattttatca ttgatgtgga ttgtgtgacg acggcaaaaa
      901 agaattactt tacacatgac tatcacacct gctcccctcc cttctccttt caatcggtca
      961 acttcccaac cgctcctcga aaatatttca gttgaatcct ctactacgta ccccttactt
     1021 aaatttactt tctctctccc catcaatgcg ccgttgttct gaagttgtat tattgatggg
     1081 agatgattgg cactgcacca gacctggcca aaagccacca caggtggcca catactcttg
     1141 catgggtata ctctaccgag gcaaagatta aagtgcacgt cccctgcccc tctctctccc
     1201 tacttccttc agtctcagtg gttccgtgcc cgttcagcac tacatcgccc tgcccgctgt
     1261 tccttctcaa acagccaccc atcccaaccc acatacacag aaacaacctt tcctacacat
     1321 tttcccaagt cttctcttca ctattctaca ctaatacacc accgtgatat gccggttatc
     1381 tagatgtcct ttctcctttc tcatcctttc tttcccatgg tacccaggag tcccattcgc
     1441 catgccgaag catgttgccc agccggcgcc agcgaggagg ctgggaccat gccggccaaa
     1501 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct
     1561 atttctagct ctaaaacgaa gagccaaggc ctgctcttca tcgattctgt ggatggaggg
     1621 agggaaggag ggggggagtg agtgagtcga gagaacgacg actggggcaa agagaggatg
     1681 acgccactgc aacaaaagac agggaagaat agaggctgtt ggaaaacagg aacacagtga
     1741 agaatagagg ctgcagggta acaggcacat ttatcgcaga gaggcgggcg gcaggggcga
     1801 tacgtaaaga gacccctaaa cttctccgcg tcatccgttc acgctacctc cttccctccc
     1861 tttactccca ttgatcacca aggcagcgcc cgcagccacc agcagccacc gtcctcagtg
     1921 tcacagctag acgcgtcccc atcaggcagc agaccaccag aatatgcgct ttcgcttccc
     1981 atcatatccc tccctcctcc ccttctattc ctcatcatgc atcatttctt tctccttgct
     2041 gcatgcccct cttcttacct tggctaagat ctaccggagg aagaagtctt caacgataga
     2101 taaatgcctg tatcgtgatg gttgtttgaa tggtggaagt gagccaggta ttgagaacga
     2161 ggggcgcaag ggtgcgtgga agaaggcagg gagaggcgag gcgaaggaga agggcatgtg
     2221 gtgggaacga agcctccaca ccacagccac ccatgctttt ctctgttcga gttcccgggt
     2281 tctactctca cacactacac gtgcacagaa aacattcacg cacaacagag atacagaggg
     2341 atccgtcgag atggtctaca cgtcctaatg tcccctgctt tcgaagccca gagccatcgc
     2401 tttctgtttt tatccattcg caacacatcc tgttctcgcg acctctcctc ttcctggcca
     2461 ccatcgcttc tccgtgccat gccctgtaca tccttccctc cctcgttgtc taccacacta
     2521 atcatgcgct ctgcagcaga cactcggcta ctatttgtgt cgtacggatt gaggggaggg
     2581 aggggaaggg aggcaagaag taaatcggtg gtcaacaaca gacgcggtct ccccaccctc
     2641 cctcattcct ttcccctcct ataatgagcg aagaaagcca tggacaccga gtcacccact
     2701 ctcctaatcc accaccacca gggcactaac ggactcacgc tcaccaaact cggtaacaat
     2761 atccactgca cacagataca cacgcaaccc tcgagcatgc ccaagaaaaa gcggaaggtg
     2821 gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg ggccgtcatt
     2881 acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac
     2941 agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac ggccgaagcc
     3001 acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg gatctgctac
     3061 ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt ccataggctg
     3121 gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat ctttggcaat
     3181 atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct gaggaagaag
     3241 cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct ggcgcatatg
     3301 atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa cagcgatgtc
     3361 gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga gaacccgatc
     3421 aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg
     3481 ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt tggtaatctt
     3541 atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct ggccgaagat
     3601 gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct gctggcccag
     3661 atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga cgccattctg
     3721 ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag cgctagtatg
     3781 atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct tgtcagacag
     3841 caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga
     3901 tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc catcttggaa
     3961 aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct gttgcgcaaa
     4021 cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga actgcacgct
     4081 atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga aaagattgag
     4141 aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg aaattccaga
     4201 ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt cgaggaagtc
     4261 gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt tgataaaaat
     4321 ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt cacagtttat
     4381 aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc attcctgtct
     4441 ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa agttaccgtg
     4501 aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc
     4561 ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct gaaaatcatt
     4621 aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga cattgtcctc
     4681 acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac ttacgctcat
     4741 ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg atgggggcgg
     4801 ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac aatcctggat
     4861 tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca tgatgactct
     4921 ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga cagtcttcac
     4981 gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt
     5041 aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc
     5101 gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg
     5161 aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt
     5221 gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac
     5281 atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc
     5341 gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat
     5401 aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat
     5461 tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact
     5521 aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt
     5581 gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc
     5641 aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag
     5701 ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac
     5761 caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat
     5821 cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg
     5881 atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat
     5941 attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca
     6001 cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg
     6061 acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag
     6121 accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca
     6181 cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac
     6241 agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag
     6301 gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt
     6361 ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac
     6421 tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag
     6481 aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac
     6541 tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa
     6601 cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc
     6661 ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc
     6721 atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct
     6781 gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag
     6841 gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac
     6901 ctctctcagc tcggtggaga cagcagggct gacgggtccg gcagcgggtc cgttaactac
     6961 ccctatgacg tgccggacta cgctagcccg aaaaagaaga ggaaggtctg acaattggaa
     7021 agatccaaga gagacgagta gagatttttt ttttgggatt gatgtttgtc gttctttgag
     7081 ttgtcgtcga gttacgcctt ttgtaagaat gttccgcagg agaggaggag gatgggcatg
     7141 agtgagggtg agagggcttg cccgcttttt tttttaaaaa cgctgaagac gtggttgtca
     7201 aacaaacccc ccatagaaac gattttgtta cggtgcggtc cagacgtcac ttgaatggct
     7261 ccgcggaaag gccagggagg gaagggggga gggaggaaac atgaaacatg ttgaacggct
     7321 caacagggtt tgggggacaa gagaggtagc gccctgatgg actgctccct cccctccttt
     7381 ccctcaatgt ctcattcatc catgcttccc ccttctctct ctcccctccg ttccatcccc
     7441 cgcgggcgtg gtagtggcgt gatgggatcc actaaaatgt acgtgtaaga aaagccggtg
     7501 agcttacgct tttgtgaaag tgggagtacg agtgttgtgt gtgtgtgtag tggtttcaga
     7561 ccccagacag aggcgaagca gaaaaagcag acgatgaaga cgacgaagaa atgagcagtc
     7621 tatttttatc gtggaaacag aagaggtgat atcgtctcct gcaggcatgc aagctgatcc
     7681 actagaggcc atggcggccg cactagtgga tcccccggga tgcatacttg gcctaggaag
     7741 atggagtgga tggaggagga ggcgagcgta gcagcaagcg tgagttatac agccaggcac
     7801 atgtcgcaat ccttcggtct cgggcttaaa atccacgcac taatcacgct gggccatgca
     7861 aagagcaatg ccgaggccca ccacacaaaa cgctgtgtcg cgcgttgcgg cctgaagctt
     7921 catacttctt agtcgccgcc aaaagggctc gagagacgag acccgttggc atgaccgatg
     7981 ttgttcgacg cggtttgctt cgtcacagtc gacgtgattc aggaatctgg agcctgcaga
     8041 tcattttttt cagcctgata tcgttctttt ccactgagaa ccatcagacc accttttctt
     8101 ccattgtgtg aaggagtagg agttgccgtg ctgctttgtg ggagacatct gcgatggtga
     8161 ccagcctccc gtcgtctggt cgacgtgacg agcctcttca ctgttcttcg acggagagac
     8221 gcaagcgaga cggctctaga ccttttggac acgcattctg tgtgtgaact agtggacagt
     8281 gataccacgt ctgaaagctc accactgccc atggtgcagc tacttgtcac aaagttttga
     8341 ctccgtcggt atcaccattc gcgctcgtgt gcctggttgt tccgccacgc cggcctgccc
     8401 cggggcgggg caatattcta aaatctcacg caaaacaccg cacttacccc tcacacatat
     8461 tcgtgataga ccaccaccaa tctcagcccg catcaacaca gtcgagggcc ccggggggca
     8521 ataagatatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
     8581 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
     8641 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta
     8701 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct
     8761 tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
     8821 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
     8881 catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
     8941 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca
     9001 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct
     9061 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga
     9121 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
     9181 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
     9241 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
     9301 atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt
     9361 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg
     9421 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
     9481 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
     9541 ggcaaaggaa tagagtagat gccgaccgga gtccgcaaaa atcaccagtc tctctctaca
     9601 aatctatctc tctctatttt tctccagaat aatgtgtgag tagttcccag ataagggaat
     9661 tagggttctt atagggtttc gctcatgtgt tgagcatata agaaaccctt agtatgtatt
     9721 tgtatttgta aaatacttct atcaataaaa tttctaattc ctaaaaccaa aatccagtga
     9781 ccagcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     9841 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     9901 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     9961 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
    10021 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
    10081 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
    10141 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
    10201 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
    10261 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
    10321 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
    10381 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
    10441 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
    10501 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
    10561 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
    10621 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
    10681 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
    10741 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
    10801 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
    10861 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
    10921 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
    10981 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
    11041 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
    11101 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
    11161 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
    11221 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
    11281 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
    11341 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
    11401 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
    11461 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
    11521 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
    11581 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
    11641 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
    11701 gtgccacctg atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
    11761 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
    11821 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
    11881 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
    11941 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
    12001 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
    12061 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
    12121 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
    12181 acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc aggctgcgca
    12241 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
    12301 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
    12361 aaacgacggc cagtgaattg taatacgact cactata
//
