LOCUS       sequence-309704-       13177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9 and BFP in a lentiviral vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13177)
  AUTHORS   Budagyan K, Chernoff J
  TITLE     A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell 
            Background.
  JOURNAL   Methods Mol Biol. 2021;2262:323-334. doi: 
            10.1007/978-1-0716-1190-6_20.
  PUBMED    33977487
REFERENCE   2  (bases 1 to 13177)
  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: "Methods Mol
            Biol."; date: "2021"; volume: "2262:323-334. doi"; pages: " 
            10.1007/978-1-0716-1190-6_20"
COMMENT     Sequence Label: pLentiCas9-P2A-BFP
FEATURES             Location/Qualifiers
     source          1..13177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /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    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2413..2624
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2643..2652
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2649..6752
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2649..6752
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6753..6800
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6801..6824
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6834..6890
                     /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(6891..6893,6894..7607)
                     /codon_start=1
                     /product="blue variant of GFP"
                     /label=BFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6891..6893,6894..7607)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(7194..7213)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7541..7562
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    7632..8220
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7685..7705)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8103..8114)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8223..8239)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8224..8240)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8745..8925
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(8951..8968)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8957..9181
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9227..9655
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9314..9333)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9524..9545
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9664..9684)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9669..9998
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9849..9984
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9911..9930
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10046..10093
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10112..10486
                     /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    10616..10737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10653..10672)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10707..10726
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10786..10802)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10786..10802)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10799..10821)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10810..10826
                     /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(10834..10840,10841..10858,10859..10864))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10879..10900
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11017..11034)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11188..11776)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11268..11287)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11947..12738,12739..12807))
                     /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     12570..12589
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12808..12912)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12987..13006)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttagctagct aggtcttgaa aggagtggga attggctccg
     2401 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     2461 tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     2521 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     2581 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg ttctagagcg
     2641 ctgccaccat ggacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct
     2701 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
     2761 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
     2821 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
     2881 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
     2941 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
     3001 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
     3061 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     3121 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     3181 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     3241 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     3301 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     3361 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     3421 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     3481 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     3541 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     3601 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     3661 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     3721 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     3781 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     3841 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     3901 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     3961 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     4021 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     4081 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
     4141 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     4201 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     4261 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     4321 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     4381 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     4441 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     4501 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     4561 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     4621 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     4681 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     4741 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     4801 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     4861 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     4921 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     4981 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     5041 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     5101 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     5161 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     5221 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     5281 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     5341 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     5401 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     5461 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     5521 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     5581 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     5641 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     5701 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     5761 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     5821 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     5881 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     5941 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     6001 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     6061 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     6121 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     6181 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     6241 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     6301 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     6361 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     6421 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     6481 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     6541 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     6601 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     6661 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     6721 cacggatcga cctgtctcag ctgggaggcg acaagcgacc tgccgccaca aagaaggctg
     6781 gacaggctaa gaagaagaaa gattacaaag acgatgacga taagggatcc ggcgcaacaa
     6841 acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg gtgagcaagg
     6901 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     6961 gccacaagtt cagcgtgtct ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     7021 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     7081 tgagccacgg ggtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     7141 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     7201 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     7261 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     7321 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggcga
     7381 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     7441 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     7501 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     7561 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagaa ttcgatatca
     7621 agcttatcgg taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     7681 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     7741 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     7801 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     7861 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     7921 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     7981 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     8041 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     8101 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     8161 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     8221 atcgataccg tcgacctcga gacctagaaa aacatggagc aatcacaagt agcaatacag
     8281 cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc
     8341 cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     8401 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata
     8461 tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag aactacacac
     8521 cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg
     8581 agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga
     8641 gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc
     8701 tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag
     8761 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     8821 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     8881 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
     8941 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     9001 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     9061 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     9121 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     9181 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     9241 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     9301 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     9361 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     9421 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     9481 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     9541 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     9601 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     9661 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     9721 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     9781 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     9841 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     9901 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     9961 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
    10021 ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat
    10081 agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt
    10141 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
    10201 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
    10261 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
    10321 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
    10381 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
    10441 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga
    10501 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
    10561 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
    10621 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
    10681 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
    10741 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
    10801 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
    10861 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
    10921 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
    10981 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
    11041 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
    11101 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
    11161 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
    11221 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
    11281 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
    11341 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
    11401 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
    11461 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
    11521 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
    11581 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
    11641 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
    11701 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
    11761 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
    11821 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
    11881 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
    11941 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
    12001 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
    12061 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
    12121 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
    12181 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
    12241 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
    12301 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
    12361 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
    12421 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
    12481 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
    12541 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
    12601 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
    12661 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
    12721 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
    12781 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
    12841 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
    12901 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
    12961 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
    13021 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
    13081 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
    13141 gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
