LOCUS       sequence-136965-       12897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pFUGW-EFSp-Cas9-P2A-Zeo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12897)
  AUTHORS   Wong AS, Choi GC, Cui CH, Pregernig G, Milani P, Adam M, Perli SD, 
            Kazer SW, Gaillard A, Hermann M, Shalek AK, Fraenkel E, Lu TK
  TITLE     Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Feb 10. pii: 201517883.
  PUBMED    26864203
REFERENCE   2  (bases 1 to 12897)
  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: "Proc Natl 
            Acad Sci U S A. 2016 Feb 10. pii: 201517883."
COMMENT     Sequence Label: pAWp30
FEATURES             Location/Qualifiers
     source          1..12897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      7..16
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             19..39
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             64..4164
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             64..4164
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4165..4212
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4228..4284
                     /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             4285..4659
                     /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"
     misc_feature    4697..5285
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4750..4770)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5168..5179)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5288..5304)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5289..5305)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5810..5990
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6016..6033)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6022..6246
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6292..6720
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6379..6398)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6589..6610
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6729..6749)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6734..7063
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6914..7049
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6976..6995
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7111..7158
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7177..7551
                     /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    7681..7802
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7718..7737)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7772..7791
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7851..7867)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7851..7867)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7864..7886)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7875..7891
                     /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(7899..7905,7906..7923,7924..7929))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7944..7965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8082..8099)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8253..8841)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8333..8352)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9012..9803,9804..9872))
                     /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     9635..9654
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9873..9977)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10052..10071)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10243..10622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        10624..10822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     10777..10797
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             10840..11020
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11067..11192
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11685..11918
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12103..12147
                     /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    12445..12562
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        12669..12880
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 ctagaggcca ccatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca
       61 gccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
      121 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
      181 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
      241 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
      301 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
      361 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
      421 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
      481 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
      541 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
      601 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
      661 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
      721 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
      781 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
      841 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
      901 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
      961 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     1021 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     1081 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     1141 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     1201 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     1261 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     1321 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     1381 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     1441 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     1501 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     1561 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     1621 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     1681 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     1741 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     1801 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     1861 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     1921 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     1981 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     2041 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     2101 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     2161 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     2221 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     2281 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     2341 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     2401 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     2461 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     2521 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     2581 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     2641 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     2701 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     2761 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     2821 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     2881 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     2941 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     3001 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     3061 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     3121 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     3181 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     3241 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     3301 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     3361 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     3421 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     3481 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     3541 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     3601 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     3661 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     3721 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     3781 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     3841 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     3901 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     3961 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     4021 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     4081 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     4141 gacctgtctc agctgggagg cgacaagcgt cctgctgcta ctaagaaagc tggtcaagct
     4201 aagaaaaaga aagctagcgg cagcggcgcc accaacttca gcctgctgaa gcaggccggc
     4261 gacgtggagg agaaccccgg ccccatggcc aagttgacca gtgccgttcc ggtgctcacc
     4321 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
     4381 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
     4441 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
     4501 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
     4561 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
     4621 ggcaactgcg tgcacttcgt ggccgaggag caggactgat agggataaca gggtaatcga
     4681 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     4741 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     4801 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     4861 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     4921 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     4981 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     5041 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     5101 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     5161 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     5221 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     5281 cccgcatcga taccgtcgac ctcgagacct agaaaaacat ggagcaatca caagtagcaa
     5341 tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg
     5401 ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct
     5461 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     5521 agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta
     5581 cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc
     5641 agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc
     5701 tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag
     5761 ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg ggtctctctg
     5821 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     5881 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     5941 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt
     6001 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     6061 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     6121 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     6181 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     6241 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     6301 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     6361 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     6421 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     6481 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     6541 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     6601 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     6661 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     6721 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     6781 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     6841 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     6901 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     6961 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     7021 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     7081 tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc
     7141 ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt
     7201 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg
     7261 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg
     7321 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg
     7381 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac
     7441 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg
     7501 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta
     7561 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     7621 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     7681 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     7741 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     7801 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     7861 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     7921 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     7981 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     8041 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     8101 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     8161 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     8221 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     8281 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     8341 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     8401 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     8461 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     8521 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     8581 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     8641 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     8701 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     8761 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     8821 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     8881 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     8941 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     9001 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     9061 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     9121 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     9181 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     9241 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     9301 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     9361 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     9421 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     9481 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     9541 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     9601 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     9661 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     9721 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     9781 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     9841 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     9901 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     9961 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
    10021 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
    10081 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
    10141 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
    10201 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
    10261 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
    10321 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
    10381 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
    10441 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
    10501 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
    10561 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
    10621 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
    10681 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
    10741 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
    10801 ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag
    10861 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
    10921 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
    10981 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
    11041 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
    11101 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
    11161 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
    11221 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
    11281 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
    11341 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
    11401 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
    11461 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
    11521 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
    11581 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
    11641 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
    11701 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
    11761 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
    11821 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
    11881 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
    11941 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
    12001 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
    12061 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
    12121 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
    12181 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
    12241 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
    12301 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
    12361 gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag
    12421 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
    12481 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
    12541 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt
    12601 aattagggat aacagggtaa tggatccgcg aaaagtccgg aattcgagtg gctccggtgc
    12661 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
    12721 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
    12781 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
    12841 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgggat
//
