LOCUS       sequence-155362-       13122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct for building stable cell lines expressing Cas9 
            in the presence of TNFa via puromycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13122)
  AUTHORS   Perli SD, Cui CH, Lu TK
  TITLE     Continuous genetic recording with self-targeting CRISPR-Cas in human
            cells.
  JOURNAL   Science. 2016 Aug 18. pii: aag0511.
  PUBMED    27540006
REFERENCE   2  (bases 1 to 13122)
  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: "Science. 
            2016 Aug 18. pii: aag0511."
COMMENT     Sequence Label: Construct 33 - NFKBRp_Cas9_3xNLS_p2a-puroR
FEATURES             Location/Qualifiers
     source          1..13122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             174..218
                     /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    516..633
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     regulatory      961..970
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             967..5070
                     /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             967..5070
                     /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             5071..5118
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5143..5163
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5170..5190
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5209..5265
                     /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             5266..5862
                     /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     5759..5779
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5890..6478
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5943..5963)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6361..6372)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6481..6497)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6482..6498)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7003..7183
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(7209..7226)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7215..7439
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7485..7913
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7572..7591)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7782..7803
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7922..7942)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7927..8256
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8107..8242
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8169..8188
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        8304..8351
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8370..8744
                     /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    8874..8995
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8911..8930)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8965..8984
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9044..9060)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9044..9060)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9057..9079)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9068..9084
                     /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(9092..9098,9099..9116,9117..9122))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9137..9158
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9275..9292)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9446..10034)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9526..9545)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10205..10996,10997..11065))
                     /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     10828..10847
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11066..11170)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(11245..11264)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        11436..11815
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11817..12015
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11970..11990
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             12033..12213
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    12260..12385
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12878..13111
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
       61 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
      121 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
      181 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
      241 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
      301 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
      361 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
      421 gtggagagag agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc
      481 caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
      541 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
      601 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
      661 ccagtttggt taattaatgg ctccgaattc gcccttcagg tccgaggttc tagacaggtg
      721 ccagaacatt tctctatcga taggtaccgg ggactttccg ggaatttccg gggactttcc
      781 gggaatttcc gggaatttcc ggggactttc cgggaatttc cggggacttt ccgggaattt
      841 ccagatctgg cctcggcggc caagcttgct agcggggggc tataaaaggg ggtgggggcg
      901 ttcgtcctca ctctagttct gcgatctaag taagcttggc attaccggtc gccaacgcgt
      961 gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     1021 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     1081 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     1141 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     1201 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     1261 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     1321 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     1381 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     1441 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     1501 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     1561 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     1621 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     1681 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     1741 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     1801 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     1861 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     1921 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     1981 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     2041 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     2101 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     2161 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     2221 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     2281 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     2341 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     2401 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     2461 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     2521 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     2581 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     2641 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     2701 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     2761 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     2821 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     2881 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     2941 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     3001 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     3061 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     3121 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     3181 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     3241 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     3301 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     3361 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     3421 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     3481 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     3541 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     3601 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     3661 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     3721 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     3781 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     3841 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     3901 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     3961 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     4021 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     4081 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     4141 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     4201 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     4261 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     4321 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     4381 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     4441 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     4501 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     4561 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     4621 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     4681 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     4741 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     4801 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     4861 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     4921 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     4981 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     5041 cggatcgacc tgtctcagct gggaggcgac aagcgtcctg ctgctactaa gaaagctggt
     5101 caagctaaga aaaagaaagc tagcggcagc ggcgccggat ccccaaagaa gaaaaggaag
     5161 gttgaagacc ccaagaaaaa gaggaaggtg ataagcgctg gaagcggagc tactaacttc
     5221 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctaccga gtacaagccc
     5281 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg
     5341 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc
     5401 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     5461 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     5521 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     5581 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     5641 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     5701 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacatc cgcgccccgc
     5761 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     5821 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gataagaatt cgatatcaag
     5881 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     5941 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     6001 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     6061 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     6121 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     6181 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     6241 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     6301 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     6361 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     6421 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     6481 cgataccgtc gacctcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca
     6541 gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca
     6601 gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     6661 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc
     6721 cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca
     6781 gggccaggga tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag
     6841 caagagaagg tagaagaagc caatgaagga gagaacaccc gcttgttaca ccctgtgagc
     6901 ctgcatggga tggatgaccc ggagagagaa gtattagagt ggaggtttga cagccgccta
     6961 gcatttcatc acatggcccg agagctgcat ccggactgta ctgggtctct ctggttagac
     7021 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     7081 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     7141 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg
     7201 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     7261 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     7321 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     7381 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     7441 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     7501 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     7561 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     7621 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     7681 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     7741 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     7801 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     7861 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     7921 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     7981 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     8041 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     8101 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     8161 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     8221 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     8281 attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag
     8341 tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc
     8401 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     8461 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     8521 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     8581 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     8641 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     8701 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt
     8761 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     8821 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     8881 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     8941 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     9001 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     9061 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     9121 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     9181 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     9241 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     9301 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     9361 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     9421 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     9481 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     9541 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     9601 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     9661 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     9721 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     9781 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     9841 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     9901 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     9961 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
    10021 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
    10081 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
    10141 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
    10201 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
    10261 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
    10321 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
    10381 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
    10441 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
    10501 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
    10561 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
    10621 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
    10681 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
    10741 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
    10801 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
    10861 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
    10921 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
    10981 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
    11041 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
    11101 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
    11161 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
    11221 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
    11281 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
    11341 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
    11401 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
    11461 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
    11521 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
    11581 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
    11641 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
    11701 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
    11761 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
    11821 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
    11881 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
    11941 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
    12001 ggtctatata agcagcgcgt tttgcctgta ctgggtctct ctggttagac cagatctgag
    12061 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
    12121 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
    12181 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
    12241 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
    12301 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
    12361 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
    12421 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
    12481 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
    12541 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
    12601 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
    12661 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
    12721 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
    12781 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
    12841 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
    12901 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
    12961 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
    13021 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
    13081 tggaaagata cctaaaggat caacagctcc tggggatttg gg
//
