LOCUS       sequence-424544-       14914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of nCas9/RT-P2A-Puro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14914)
  AUTHORS   Ren X, Yang H, Nierenberg JL, Sun Y, Chen J, Beaman C, Pham T, 
            Nobuhara M, Takagi MA, Narayan V, Li Y, Ziv E, Shen Y
  TITLE     High-throughput PRIME-editing screens identify functional DNA 
            variants in the human genome.
  JOURNAL   Mol Cell. 2023 Dec 21;83(24):4633-4645.e9. doi: 
            10.1016/j.molcel.2023.11.021.
  PUBMED    38134886
REFERENCE   2  (bases 1 to 14914)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2023.11"; journalName: "Mol Cell"; date: 
            "2023-12-21- 21"; volume: "83"; issue: "24"; pages: "4633-4645.e9"
COMMENT     Sequence Label: LentiV2-EF1-alpha-nCas9/RT
FEATURES             Location/Qualifiers
     source          1..14914
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        13..211
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     166..186
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             229..409
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    456..581
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1074..1307
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1492..1536
                     /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    1834..1951
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2051..2262
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     2288..2307
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2288..2306
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2356..2376
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2377..4890,4891..4893,4894..6477)
                     /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             2377..6477
                     /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             8650..8697
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8698..8721
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8731..8787
                     /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             8788..9384
                     /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     9281..9301
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9400..9988
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9453..9473)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9871..9882)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10060..10293
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10319..10336)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10325..10549
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10595..11023
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10682..10701)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10892..10913
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11032..11052)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11037..11366
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11217..11352
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11279..11298
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11414..11461
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11480..11854
                     /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    11984..12105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12021..12040)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12075..12094
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12154..12170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12154..12170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12167..12189)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12178..12194
                     /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(12202..12208,12209..12226,12227..12232))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12247..12268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12385..12402)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12556..13144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12636..12655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13315..14106,14107..14175))
                     /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     13938..13957
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14176..14280)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(14355..14374)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(14546..14914,1..11)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
       61 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      121 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      181 ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg
      241 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      301 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      361 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      421 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      481 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      541 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      601 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      661 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      721 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      781 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
      841 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
      901 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
      961 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1021 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1081 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1141 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1201 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1261 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1321 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1381 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1441 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1501 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1561 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1621 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1681 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     1741 ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca
     1801 attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     1861 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     1921 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     1981 agtttggtta attaagaatt cgctagctag gtcttgaaag gagtgggaat tggctccggt
     2041 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2101 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2161 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2221 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagaagcgc
     2281 tggccgctaa tacgactcac tatagggaga gccgccacca tgaaacggac agccgacgga
     2341 agcgagttcg agtcaccaaa gaagaagcgg aaagtcgaca agaagtacag catcggcctg
     2401 gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
     2461 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
     2521 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
     2581 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
     2641 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
     2701 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
     2761 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
     2821 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
     2881 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
     2941 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     3001 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     3061 cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc
     3121 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     3181 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     3241 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     3301 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     3361 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     3421 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     3481 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     3541 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     3601 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     3661 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     3721 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     3781 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     3841 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     3901 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     3961 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     4021 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     4081 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     4141 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     4201 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     4261 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     4321 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     4381 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     4441 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     4501 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     4561 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     4621 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     4681 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     4741 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     4801 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     4861 atcaaccggc tgtccgacta cgatgtggac gctatcgtgc ctcagagctt tctgaaggac
     4921 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     4981 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     5041 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     5101 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     5161 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     5221 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     5281 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     5341 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     5401 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     5461 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     5521 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     5581 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     5641 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     5701 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     5761 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     5821 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     5881 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     5941 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     6001 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     6061 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     6121 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     6181 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     6241 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     6301 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     6361 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     6421 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggtgactct
     6481 ggaggatcta gcggaggatc ctctggcagc gagacaccag gaacaagcga gtcagcaaca
     6541 ccagagagca gtggcggcag cagcggcggc agcagcaccc taaatataga agatgagtat
     6601 cggctacatg agacctcaaa agagccagat gtttctctag ggtccacatg gctgtctgat
     6661 tttcctcagg cctgggcgga aaccgggggc atgggactgg cagttcgcca agctcctctg
     6721 atcatacctc tgaaagcaac ctctaccccc gtgtccataa aacaataccc catgtcacaa
     6781 gaagccagac tggggatcaa gccccacata cagagactgt tggaccaggg aatactggta
     6841 ccctgccagt ccccctggaa cacgcccctg ctacccgtta agaaaccagg gactaatgat
     6901 tataggcctg tccaggatct gagagaagtc aacaagcggg tggaagacat ccaccccacc
     6961 gtgcccaacc cttacaacct cttgagcggg ctcccaccgt cccaccagtg gtacactgtg
     7021 cttgatttaa aggatgcctt tttctgcctg agactccacc ccaccagtca gcctctcttc
     7081 gcctttgagt ggagagatcc agagatggga atctcaggac aattgacctg gaccagactc
     7141 ccacagggtt tcaaaaacag tcccaccctg tttaatgagg cactgcacag agacctagca
     7201 gacttccgga tccagcaccc agacttgatc ctgctacagt acgtggatga cttactgctg
     7261 gccgccactt ctgagctaga ctgccaacaa ggtactcggg ccctgttaca aaccctaggg
     7321 aacctcgggt atcgggcctc ggccaagaaa gcccaaattt gccagaaaca ggtcaagtat
     7381 ctggggtatc ttctaaaaga gggtcagaga tggctgactg aggccagaaa agagactgtg
     7441 atggggcagc ctactccgaa gacccctcga caactaaggg agttcctagg gaaggcaggc
     7501 ttctgtcgcc tcttcatccc tgggtttgca gaaatggcag cccccctgta ccctctcacc
     7561 aaaccgggga ctctgtttaa ttggggccca gaccaacaaa aggcctatca agaaatcaag
     7621 caagctcttc taactgcccc agccctgggg ttgccagatt tgactaagcc ctttgaactc
     7681 tttgtcgacg agaagcaggg ctacgccaaa ggtgtcctaa cgcaaaaact gggaccttgg
     7741 cgtcggccgg tggcctacct gtccaaaaag ctagacccag tagcagctgg gtggccccct
     7801 tgcctacgga tggtagcagc cattgccgta ctgacaaagg atgcaggcaa gctaaccatg
     7861 ggacagccac tagtcattct ggccccccat gcagtagagg cactagtcaa acaacccccc
     7921 gaccgctggc tttccaacgc ccggatgact cactatcagg ccttgctttt ggacacggac
     7981 cgggtccagt tcggaccggt ggtagccctg aacccggcta cgctgctccc actgcctgag
     8041 gaagggctgc aacacaactg ccttgatatc ctggccgaag cccacggaac ccgacccgac
     8101 ctaacggacc agccgctccc agacgccgac cacacctggt acacggatgg aagcagtctc
     8161 ttacaagagg gacagcgtaa ggcgggagct gcggtgacca ccgagaccga ggtaatctgg
     8221 gctaaagccc tgccagccgg gacatccgct cagcgggctg aactgatagc actcacccag
     8281 gccctaaaga tggcagaagg taagaagcta aatgtttata ctgatagccg ttatgctttt
     8341 gctactgccc atatccatgg agaaatatac agaaggcgtg ggtggctcac atcagaaggc
     8401 aaagagatca aaaataaaga cgagatcttg gccctactaa aagccctctt tctgcccaaa
     8461 agacttagca taatccattg tccaggacat caaaagggac acagcgccga ggctagaggc
     8521 aaccggatgg ctgaccaagc ggcccgaaag gcagccatca cagagactcc agacacctct
     8581 accctcctca tagaaaattc atcaccctct ggcggctcaa aaagaaccgc cgacggcagc
     8641 gaatttgaga agcgacctgc cgccacaaag aaggctggac aggctaagaa gaagaaagat
     8701 tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc
     8761 ggagatgtcg aagagaatcc tggaccgacc gagtacaagc ccacggtgcg cctcgccacc
     8821 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     8881 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     8941 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     9001 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     9061 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     9121 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     9181 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     9241 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     9301 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     9361 atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa
     9421 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     9481 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     9541 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     9601 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     9661 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     9721 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     9781 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     9841 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     9901 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     9961 cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag
    10021 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
    10081 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
    10141 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
    10201 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
    10261 gaccctttta gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc
    10321 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
    10381 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
    10441 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
    10501 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
    10561 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
    10621 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
    10681 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
    10741 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
    10801 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
    10861 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
    10921 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
    10981 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
    11041 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
    11101 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
    11161 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
    11221 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
    11281 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
    11341 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
    11401 ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
    11461 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
    11521 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
    11581 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
    11641 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
    11701 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
    11761 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
    11821 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac
    11881 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
    11941 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
    12001 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
    12061 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
    12121 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
    12181 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
    12241 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
    12301 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
    12361 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
    12421 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
    12481 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
    12541 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
    12601 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
    12661 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
    12721 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
    12781 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
    12841 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
    12901 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
    12961 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
    13021 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
    13081 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
    13141 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
    13201 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
    13261 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
    13321 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
    13381 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
    13441 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
    13501 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
    13561 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
    13621 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
    13681 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
    13741 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
    13801 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
    13861 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
    13921 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
    13981 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
    14041 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
    14101 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
    14161 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
    14221 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
    14281 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
    14341 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
    14401 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
    14461 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
    14521 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
    14581 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
    14641 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
    14701 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
    14761 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
    14821 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
    14881 cctacttggc agtacatcta cgtattagtc atcg
//
