LOCUS       sequence-256796-        9798 bp    DNA     circular SYN 15-APR-2025
DEFINITION  NKX3-1 knock out.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9798)
  TITLE     Lian Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 9798)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pYJ39-PB-CRISPR-NKX3.1-E2-gRNA2
FEATURES             Location/Qualifiers
     source          1..9798
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..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     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        998..1246
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     998..1018
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1169..1188
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1267..1342
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1390..1507
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1576..1787
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1807..1816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1816..1839
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1846..1866
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1891..5991
                     /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             1891..5991
                     /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             5992..6039
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6055..6111
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6112..6711
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6112..6131)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6608..6628
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    6727..7315
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6780..6800)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7198..7209)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   7497..7559
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7610..7630)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7610..7628)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7649..7665)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7649..7665)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7662..7684)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7673..7689
                     /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(7697..7703,7704..7721,7722..7727))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7742..7763
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7880..7897)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8051..8639)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8131..8150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8810..9601,9602..9670))
                     /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     9433..9452
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9671..9775)
                     /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 gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcgag ggcctatttc ccatgattcc
     1021 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     1081 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     1141 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     1201 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgtta tacacggaga
     1261 ccaggggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     1321 aaaagtggca ccgagtcggt gcttttttga attctagatc ttgagacaaa tggcagtatt
     1381 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     1441 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     1501 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct
     1561 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     1621 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     1681 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     1741 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg
     1801 ggatccgcca ccatggatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
     1861 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
     1921 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     1981 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     2041 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     2101 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     2161 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     2221 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     2281 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     2341 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     2401 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     2461 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     2521 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     2581 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     2641 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     2701 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     2761 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     2821 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     2881 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     2941 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     3001 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     3061 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     3121 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     3181 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     3241 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     3301 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     3361 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     3421 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     3481 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     3541 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     3601 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     3661 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     3721 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     3781 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     3841 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     3901 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     3961 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     4021 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     4081 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     4141 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     4201 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     4261 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     4321 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     4381 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     4441 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     4501 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     4561 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     4621 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     4681 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     4741 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     4801 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     4861 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     4921 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     4981 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     5041 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     5101 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     5161 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     5221 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     5281 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     5341 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     5401 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     5461 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     5521 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     5581 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     5641 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     5701 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     5761 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     5821 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     5881 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     5941 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caagcgtcct
     6001 gctgctacta agaaagctgg tcaagctaag aaaaagaaag ctagcggcag cggcgccacc
     6061 aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc catgaccgag
     6121 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     6181 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     6241 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     6301 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     6361 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     6421 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     6481 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     6541 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     6601 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     6661 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     6721 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     6781 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     6841 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     6901 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     6961 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     7021 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     7081 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     7141 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     7201 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     7261 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcactag
     7321 ttaaaagttt tgttacttta tagaagaaat tttgagtttt tgtttttttt taataaataa
     7381 ataaacataa ataaattgtt tgttgaattt attattagta tgtaagtgta aatataataa
     7441 aacttaatat ctattcaaat taataaataa acctcgatat acagaccgat aaaacacatg
     7501 cgtcaatttt acgcatgatt atctttaacg tacgtcacaa tatgattatc tttctagggt
     7561 taatctagta tacgcgtatg cggccgctta attaatccag cttttgttcc ctttagtgag
     7621 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     7681 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     7741 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     7801 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     7861 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     7921 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     7981 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     8041 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     8101 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     8161 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     8221 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     8281 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     8341 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     8401 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     8461 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     8521 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     8581 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     8641 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     8701 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     8761 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     8821 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     8881 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     8941 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     9001 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     9061 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     9121 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     9181 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     9241 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     9301 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     9361 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     9421 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     9481 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     9541 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     9601 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     9661 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     9721 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     9781 ttccccgaaa agtgccac
//
