LOCUS       sequence-361798-       14972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single EF1a-driven PE2-P2A-GFP (Addgene #132776) and PaqCI 
            restriction sites for pegRNA cloning. Lentiviral backbone and AmpR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14972)
  AUTHORS   Dirkx N, Weuring WJ, De Vriendt E, Smal N, van de Vondervoort J, van
            't Slot R, Koetsier M, Zonnekein N, De Pooter T, Weckhuysen S, 
            Koeleman BPC
  TITLE     Increased prime edit rates in KCNQ2 and SCN1A via single nicking 
            all-in-one plasmids.
  JOURNAL   BMC Biol. 2023 Jul 13;21(1):156. doi: 10.1186/s12915-023-01646-7.
  PUBMED    37443005
REFERENCE   2  (bases 1 to 14972)
  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: "BMC Biol.";
            date: "2023-07-13"; volume: "21(1):156. doi"; pages: " 
            10.1186/s12915-023-01646-7"
COMMENT     Sequence Label: pLenti_Ef1a:PE2-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..14972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(285..304)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     632..653
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             708..728
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             737..754
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    792..1380
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(845..865)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1263..1274)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1587..2220
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(2348..2364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2348..2364)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2361..2383)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2372..2388
                     /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(2396..2402,2403..2420,2421..2426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2441..2462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2579..2596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2750..3338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2830..2849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3509..4300,4301..4369))
                     /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     4132..4151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4370..4474)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4522..4656
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4559..4578)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4613..4632
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             4824..5457
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5504..5629
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6126..6359
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6544..6588
                     /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    6851..6966
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        7256..8438
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          7487..8429
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     8386..8406
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     8502..8521
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8502..8520
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             8570..8590
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(8591..11104,11105..11107,11108..12691)
                     /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             8591..12691
                     /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             14864..14884
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             14894..14950
                     /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             join(14951..14953,14954..14956,14957..14972,1..695)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(14951..14953,14954..14956,14957..14972,1..695)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
ORIGIN
        1 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
       61 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
      121 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctatg
      181 gagtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
      241 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
      301 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
      361 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
      421 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
      481 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
      541 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
      601 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
      661 ccgggatcac tctcggcatg gacgagctgt acaagtctgg tggttctccc aagaagaaga
      721 ggaaagtcta accggtcatc atcaccatca ccattgagtt ttaccaccgg tggaattcac
      781 gcgtctggaa caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
      841 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
      901 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
      961 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     1021 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     1081 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     1141 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc
     1201 catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     1261 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     1321 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     1381 ctggaattaa ttctgcagtc gagacctaga aaaacatgga gcaatcacaa gtagcaatac
     1441 agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt
     1501 tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     1561 ccacttttta aaagaaaaga ggggactgga agggctaatt cactcccaac gaagacaaga
     1621 tatccttgat ctgtggatct accacacaca aggctacttc cctgattagc agaactacac
     1681 accagggcca ggggtcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
     1741 tgagccagat aaggtagaag aggccaataa aggagagaac accagcttgt tacaccctgt
     1801 gagcctgcat gggatggatg acccggagag agaagtgtta gagtggaggt ttgacagccg
     1861 cctagcattt catcacgtgg cccgagagct gcatccggag tacttcaaga actgctgata
     1921 tcgagcttgc tacaagggac tttccgctgg ggactttcca gggaggcgtg gcctgggcgg
     1981 gactggggag tggcgagccc tcagatcctg catataagca gctgcttttt gcctgtactg
     2041 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     2101 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     2161 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     2221 gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     2281 agagtgagag gccttgacat tgctagcgtt taccgtcgac ctctagctag agcttggcgt
     2341 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2401 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2461 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2521 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2581 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2641 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3181 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3421 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3481 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3541 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3601 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3661 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3721 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3781 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3841 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3901 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3961 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4021 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4081 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4141 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4201 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4261 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4321 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4381 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4501 acgtcgacgg atcgggagat caacttgttt attgcagctt ataatggtta caaataaagc
     4561 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4621 tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca agctaaccaa
     4681 aatcatccca aacttcccac cccataccct attaccactg ccaattacct gtggtttcat
     4741 ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg tatttgttaa
     4801 atatgtacta caaacttagt agttggaagg gctaattcac tcccaaagaa gacaagatat
     4861 ccttgatctg tggatctacc acacacaagg ctacttccct gattagcaga actacacacc
     4921 agggccaggg gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     4981 gccagataag gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag
     5041 cctgcatggg atggatgacc cggagagaga agtgttagag tggaggtttg acagccgcct
     5101 agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact gctgatatcg
     5161 agcttgctac aagggacttt ccgctgggga ctttccaggg aggcgtggcc tgggcgggac
     5221 tggggagtgg cgagccctca gatcctgcat ataagcagct gctttttgcc tgtactgggt
     5281 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     5341 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     5401 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
     5461 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
     5521 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
     5581 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
     5641 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
     5701 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
     5761 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
     5821 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
     5881 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
     5941 caaaagtaag accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga
     6001 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     6061 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     6121 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     6181 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     6241 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     6301 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     6361 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     6421 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     6481 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     6541 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     6601 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     6661 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     6721 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     6781 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatctcga
     6841 cggtatcgcc tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     6901 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     6961 aaattttcgg gtttattaca gggacagcag agatccagtt tatcgatggt ccgatttcag
     7021 caggtgatga ggttagcagt ctcacctgcg ggcagatcga acctgcaggt tccactggga
     7081 cgattacatg catctggatc gatgagtaat tcatacaaaa ggactcgccc ctgccttggg
     7141 gaatcccagg gaccgtcgtt aaactcccac taacgtagaa cccagagatc gctgcgttcc
     7201 cgccccctca cccgcccgct ctcgtcatca ctgaggtgga gaagagcatg cgtgaggctc
     7261 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     7321 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     7381 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     7441 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
     7501 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
     7561 cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     7621 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     7681 ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     7741 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     7801 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     7861 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     7921 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     7981 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     8041 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     8101 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     8161 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     8221 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     8281 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     8341 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     8401 tggttcaaag tttttttctt ccatttcagg tgtcgtgagg atctatttcc ggtgaattgg
     8461 tttaaacgcg atcgcctcga gggatcctct agagcggccg ctaatacgac tcactatagg
     8521 gagagccgcc accatgaaac ggacagccga cggaagcgag ttcgagtcac caaagaagaa
     8581 gcggaaagtc gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
     8641 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     8701 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     8761 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     8821 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     8881 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     8941 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     9001 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     9061 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     9121 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     9181 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     9241 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     9301 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     9361 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     9421 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     9481 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     9541 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     9601 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     9661 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     9721 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     9781 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     9841 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     9901 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     9961 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
    10021 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt
    10081 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
    10141 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
    10201 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
    10261 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
    10321 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
    10381 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
    10441 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
    10501 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
    10561 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
    10621 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
    10681 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
    10741 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
    10801 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
    10861 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
    10921 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
    10981 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
    11041 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
    11101 ggacgctatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac
    11161 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
    11221 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
    11281 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
    11341 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
    11401 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
    11461 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
    11521 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
    11581 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
    11641 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
    11701 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
    11761 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
    11821 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
    11881 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
    11941 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
    12001 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
    12061 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
    12121 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
    12181 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
    12241 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
    12301 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
    12361 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
    12421 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
    12481 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
    12541 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
    12601 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
    12661 acggatcgac ctgtctcagc tgggaggtga ctctggagga tctagcggag gatcctctgg
    12721 cagcgagaca ccaggaacaa gcgagtcagc aacaccagag agcagtggcg gcagcagcgg
    12781 cggcagcagc accctaaata tagaagatga gtatcggcta catgagacct caaaagagcc
    12841 agatgtttct ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg
    12901 gggcatggga ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac
    12961 ccccgtgtcc ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca
    13021 catacagaga ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc
    13081 cctgctaccc gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga
    13141 agtcaacaag cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag
    13201 cgggctccca ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg
    13261 cctgagactc caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat
    13321 gggaatctca ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac
    13381 cctgtttaat gaggcactgc acagagacct agcagacttc cggatccagc acccagactt
    13441 gatcctgcta cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca
    13501 acaaggtact cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa
    13561 gaaagcccaa atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca
    13621 gagatggctg actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc
    13681 tcgacaacta agggagttcc tagggaaggc aggcttctgt cgcctcttca tccctgggtt
    13741 tgcagaaatg gcagcccccc tgtaccctct caccaaaccg gggactctgt ttaattgggg
    13801 cccagaccaa caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct
    13861 ggggttgcca gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc
    13921 caaaggtgtc ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa
    13981 aaagctagac ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc
    14041 cgtactgaca aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc
    14101 ccatgcagta gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat
    14161 gactcactat caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc
    14221 cctgaacccg gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga
    14281 tatcctggcc gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc
    14341 cgaccacacc tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg
    14401 agctgcggtg accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc
    14461 cgctcagcgg gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa
    14521 gctaaatgtt tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat
    14581 atacagaagg cgtgggtggc tcacatcaga aggcaaagag atcaaaaata aagacgagat
    14641 cttggcccta ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg
    14701 acatcaaaag ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg
    14761 aaaggcagcc atcacagaga ctccagacac ctctaccctc ctcatagaaa attcatcacc
    14821 ctctggcggc tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga agaagaggaa
    14881 agtcggaagc ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa
    14941 ccctggacct atggtgagca agggcgagga gc
//
