LOCUS       sequence-275160-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TOP2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Kwan SY, Sheel A, Song CQ, Zhang XO, Jiang T, Dang H, Cao Y, Ozata 
            DM, Mou H, Yin H, Weng Z, Wang XW, Xue W
  TITLE     Depletion of TRRAP induces p53-independent senescence in liver 
            cancer by downregulating mitotic genes.
  JOURNAL   Hepatology. 2019 Jun 12. doi: 10.1002/hep.30807.
  PUBMED    31188495
REFERENCE   2  (bases 1 to 13013)
  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: "Hepatology.
            2019 Jun 12. doi: 10.1002/hep.30807."
COMMENT     Sequence Label: TOP2A sgRNA1
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..200
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             218..398
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    445..570
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1063..1296
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1481..1525
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1823..1940
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1991..2239
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1991..2011
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2162..2181
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2261..2336
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2398..2609
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2628..2637
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2634..6737
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2634..6737
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6738..6785
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6786..6809
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6819..6875
                     /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             6876..7472
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7369..7389
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7488..8076
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7541..7561)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7959..7970)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8148..8381
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8407..8424)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8413..8637
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8683..9111
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8770..8789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8980..9001
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9120..9140)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9125..9454
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9305..9440
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9367..9386
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9502..9549
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9568..9942
                     /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="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    10072..10193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10109..10128)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10163..10182
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10242..10258)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10242..10258)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10255..10277)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10266..10282
                     /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(10290..10296,10297..10314,10315..10320))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10335..10356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10473..10490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10644..11232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10724..10743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11403..12194,12195..12263))
                     /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     12026..12045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12264..12368)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12443..12462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12634..13013
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat
      241 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
      301 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
      361 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
      421 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
      481 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
      541 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
      601 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
      661 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
      721 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
      781 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
      841 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
      901 cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
      961 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1021 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1081 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1141 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1201 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1261 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1321 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1381 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1441 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1501 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1561 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1621 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1681 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1741 cagagacaga tccattcgat tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac
     1801 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag
     1861 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     1921 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttaa
     1981 ttaaggtacc gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     2041 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     2101 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     2161 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     2221 gtggaaagga cgaaacaccg tgtacgctta tcctgactga gttttagagc tagaaatagc
     2281 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     2341 ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
     2401 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg
     2461 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     2521 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     2581 ttcgcaacgg gtttgccgcc agaacacagg accggttcta gagcgctgcc accatggaca
     2641 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg
     2701 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     2761 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     2821 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     2881 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     2941 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     3001 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     3061 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     3121 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     3181 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     3241 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     3301 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg
     3361 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     3421 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     3481 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     3541 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     3601 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     3661 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     3721 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     3781 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     3841 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     3901 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     3961 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     4021 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     4081 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     4141 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     4201 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     4261 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     4321 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     4381 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     4441 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     4501 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     4561 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     4621 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     4681 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     4741 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     4801 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     4861 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     4921 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     4981 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     5041 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     5101 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc
     5161 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     5221 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     5281 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     5341 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     5401 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     5461 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     5521 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     5581 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     5641 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     5701 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     5761 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     5821 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     5881 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     5941 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     6001 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     6061 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     6121 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     6181 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     6241 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     6301 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     6361 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     6421 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     6481 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     6541 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     6601 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     6661 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     6721 ctcagctggg aggcgacaag cgacctgccg ccacaaagaa ggctggacag gctaagaaga
     6781 agaaagatta caaagacgat gacgataagg gatccggcgc aacaaacttc tctctgctga
     6841 aacaagccgg agatgtcgaa gagaatcctg gaccgaccga gtacaagccc acggtgcgcc
     6901 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     6961 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     7021 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     7081 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     7141 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     7201 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag
     7261 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     7321 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     7381 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     7441 cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg
     7501 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     7561 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     7621 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     7681 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     7741 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     7801 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     7861 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     7921 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     7981 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     8041 agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta
     8101 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     8161 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     8221 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     8281 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     8341 atccctcaga cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct
     8401 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     8461 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     8521 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     8581 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     8641 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     8701 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     8761 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     8821 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     8881 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     8941 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     9001 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     9061 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     9121 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     9181 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     9241 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     9301 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     9361 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     9421 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     9481 tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta
     9541 taatacgaca aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc
     9601 accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg
     9661 gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc
     9721 gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg
     9781 gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg
     9841 ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg
     9901 gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct acgagatttc
     9961 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
    10021 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
    10081 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
    10141 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
    10201 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
    10261 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
    10321 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
    10381 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
    10441 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
    10501 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
    10561 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
    10621 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
    10681 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
    10741 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
    10801 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
    10861 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
    10921 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
    10981 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
    11041 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
    11101 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
    11161 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
    11221 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
    11281 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
    11341 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
    11401 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
    11461 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
    11521 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
    11581 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
    11641 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
    11701 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
    11761 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
    11821 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
    11881 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
    11941 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
    12001 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
    12061 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
    12121 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
    12181 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
    12241 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
    12301 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
    12361 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
    12421 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
    12481 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
    12541 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
    12601 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
    12661 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
    12721 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
    12781 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
    12841 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
    12901 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
    12961 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atg
//
