LOCUS       sequence-432865-       14992 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral plasmid to produce the prototypical CE1 construct 
            (PCV2-nCas9-EcKlenow), expressed from the EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14992)
  AUTHORS   Ferreira da Silva J, Tou CJ, King EM, Eller ML, Rufino-Ramos D, Ma 
            L, Cromwell CR, Metovic J, Benning FMC, Chao LH, Eichler FS, 
            Kleinstiver BP
  TITLE     Click editing enables programmable genome writing using DNA 
            polymerases and HUH endonucleases.
  JOURNAL   Nat Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x.
  PUBMED    39039307
REFERENCE   2  (bases 1 to 14992)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x."
COMMENT     Sequence Label: Lentiviral click editor (CE1) plasmid -
            LTR-pE...
FEATURES             Location/Qualifiers
     source          1..14992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1480..1524
                     /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    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2045..2256
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             join(2704..5217,5218..5220,5221..6804)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2704..6804
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6862..6882
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8767..8787
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8797..8853
                     /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             8854..9450
                     /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     9347..9367
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9466..10054
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9519..9539)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9937..9948)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10126..10359
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10385..10402)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10391..10615
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10661..11089
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10748..10767)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10958..10979
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11098..11118)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11103..11432
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11283..11418
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11345..11364
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11480..11527
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11546..11920
                     /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    12050..12171
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12087..12106)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12141..12160
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12220..12236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12220..12236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12233..12255)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12244..12260
                     /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(12268..12274,12275..12292,12293..12298))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12313..12334
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12451..12468)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12622..13210)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12702..12721)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13381..14172,14173..14241))
                     /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     14004..14023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14242..14346)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(14421..14440)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        14612..14991
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg aattcgctag ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt
     2041 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     2101 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     2161 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     2221 gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag cgctgccacc
     2281 atgagcccat ctaagaaaaa cggcagaagc ggacctcaac ctcacaagcg gtgggtcttt
     2341 accctgaaca accccagcga agatgagaga aagaagatcc gggacctgcc tatcagcctg
     2401 ttcgactact tcatcgtggg cgaggaagga aatgaggaag gccggacccc ccacctgcag
     2461 ggcttcgcca acttcgtgaa aaagcagacc ttcaacaagg tgaagtggta cctgggcgct
     2521 agatgccaca tcgagaaggc caagggcacc gaccagcaga acaaagagta ctgcagcaag
     2581 gaaggcaatc tgctgatgga atgtggcgcc cctagatccc agggacagag aagcggaggc
     2641 tctagcggct ccgagacacc cggcacatct gagtccgcca cccctgagag cagcggcggc
     2701 agcgacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
     2761 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     2821 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     2881 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     2941 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     3001 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     3061 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     3121 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     3181 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     3241 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     3301 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     3361 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     3421 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     3481 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     3541 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     3601 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     3661 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     3721 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     3781 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     3841 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     3901 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     3961 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     4021 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     4081 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     4141 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     4201 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     4261 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     4321 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     4381 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     4441 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     4501 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     4561 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     4621 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     4681 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     4741 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     4801 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     4861 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     4921 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     4981 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     5041 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     5101 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     5161 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggacgct
     5221 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     5281 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     5341 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     5401 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     5461 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     5521 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     5581 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     5641 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     5701 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     5761 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     5821 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     5881 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     5941 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     6001 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     6061 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     6121 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     6181 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     6241 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     6301 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     6361 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     6421 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     6481 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     6541 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     6601 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     6661 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     6721 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     6781 gacctgtctc agctgggagg tgactccggc ggaagctctg gtggcagcaa gcggaccgcc
     6841 gacggctctg aattcgagag ccctaagaag aaaagaaagg tgagcggagg ctctagcggc
     6901 ggaagcgtta tctcctacga caactacgtg accatcctgg acgaagagac actgaaggct
     6961 tggatcgcta aactggaaaa agcccctgtg ttcgccttcg ccaccgccac cgactcctta
     7021 gataacatca gcgccaacct ggtgggcctg tctttcgcca tcgagcctgg cgtggccgcc
     7081 tacatccccg tggcccacga ttacctggat gccccagatc aaatcagcag agagagagca
     7141 ctggagctcc tgaagcccct gctggaggac gagaaggccc tgaaggtggg acagaacctg
     7201 aaatacgata ggggaatcct ggctaattac ggcatcgagc tgagaggcat cgccttcgat
     7261 accatgctgg aatcttacat cctgaacagc gtggccggcc gccacgacat ggacagcctg
     7321 gctgaacggt ggctgaagca caaaaccatc accttcgaag agatcgccgg aaagggcaag
     7381 aaccagctga catttaatca gatcgcactg gaagaagccg gcagatacgc cgctgaggac
     7441 gccgacgtga cccttcagct gcacctgaag atgtggcccg atctgcagaa gcacaagggc
     7501 ccactaaacg tgttcgagaa catcgagatg cctctggtgc ccgtcctgag cagaatcgag
     7561 aggaatggcg tgaagatcga ccccaaggtg ctgcacaacc acagcgagga actgacactg
     7621 cggctggccg agctggaaaa gaaggcccac gaaatcgccg gcgaggagtt caacctgtct
     7681 agcaccaagc agctacaaac aatcctgttc gagaagcagg gcattaagcc tctgaaaaag
     7741 acacctggcg gagcccccag caccagcgaa gaagtgctgg aggaactggc gctggattac
     7801 cctctgccca aagtgatcct ggaatataga ggactggcca agctgaagtc tacatacacc
     7861 gacaagctgc ctctgatgat caaccccaag accggccggg tgcatacatc ttatcaccag
     7921 gccgtgacag ccaccggccg gctgagcagc acagatccta acctgcaaaa catcccagtg
     7981 cggaacgaag agggcagaag aatcagacag gctttcattg cccctgagga ctacgtcatc
     8041 gtgtctgctg actacagcca gatagaactg agaatcatgg cccatctgtc tcgggacaag
     8101 ggcctgctga ccgcttttgc agaaggcaag gacatccaca gagccaccgc tgccgaagtg
     8161 ttcggcctgc ctctggaaac agtgaccagc gagcaaagaa ggtccgccaa ggccattaac
     8221 ttcggcctga tctacggcat gagcgccttt ggcctggcca gacagctcaa catccctaga
     8281 aaggaggccc agaaatacat ggacctgtac ttcgagcggt accccggcgt gctggagtac
     8341 atggaacgga ccagagccca ggctaaagag cagggatacg tggaaaccct ggatggccgg
     8401 agactgtatc tgcctgacat caagagcagc aacggcgcca gaagagccgc cgctgagaga
     8461 gctgctatca acgcccctat gcagggcacc gccgccgaca tcatcaagcg ggccatgatc
     8521 gccgtggacg cctggctgca ggccgagcag cctagagtgc ggatgatcat gcaggtgcac
     8581 gacgagctgg tgtttgaggt gcacaaggac gacgtggatg ctgtggccaa gcagattcac
     8641 cagctgatgg aaaattgcac cagactggac gtgccactgc tggtcgaggt tggaagcgga
     8701 gagaattggg accaggccca ctctggcggc tcaaaaagaa ccgccgacgg cagcgaattc
     8761 gagtctccca agaagaagag gaaagtcgga tccggcgcaa caaacttctc tctgctgaaa
     8821 caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac ggtgcgcctc
     8881 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     8941 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     9001 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     9061 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     9121 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     9181 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
     9241 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     9301 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     9361 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     9421 tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat
     9481 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     9541 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     9601 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     9661 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     9721 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     9781 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     9841 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     9901 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     9961 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
    10021 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
    10081 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
    10141 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
    10201 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
    10261 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
    10321 ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga
    10381 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
    10441 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
    10501 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
    10561 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
    10621 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
    10681 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
    10741 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
    10801 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
    10861 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
    10921 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
    10981 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
    11041 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
    11101 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
    11161 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
    11221 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
    11281 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
    11341 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
    11401 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
    11461 tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata
    11521 atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac
    11581 cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga
    11641 cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc
    11701 ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga
    11761 cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc
    11821 ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc
    11881 cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga
    11941 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
    12001 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
    12061 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
    12121 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
    12181 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
    12241 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
    12301 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
    12361 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
    12421 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
    12481 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
    12541 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
    12601 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
    12661 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
    12721 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
    12781 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
    12841 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
    12901 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
    12961 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
    13021 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
    13081 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
    13141 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
    13201 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
    13261 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
    13321 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
    13381 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
    13441 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
    13501 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
    13561 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
    13621 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
    13681 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
    13741 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
    13801 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
    13861 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
    13921 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
    13981 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
    14041 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
    14101 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
    14161 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
    14221 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
    14281 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
    14341 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
    14401 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
    14461 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
    14521 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
    14581 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
    14641 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
    14701 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
    14761 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
    14821 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
    14881 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
    14941 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gg
//
