LOCUS       sequence-308012-       14681 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral backbone for cloning and expression of U6 driven gRNAs 
            with Esp3I/BsmBI cloning sites, puromycin selection and mClover3 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14681)
  AUTHORS   Aregger M, Lawson KA, Billmann M, Costanzo M, Tong AHY, Chan K, 
            Rahman M, Brown KR, Ross C, Usaj M, Nedyalkova L, Sizova O, Habsid 
            A, Pawling J, Lin ZY, Abdouni H, Wong CJ, Weiss A, Mero P, Dennis 
            JW, Gingras AC, Myers CL, Andrews BJ, Boone C, Moffat J
  TITLE     Systematic mapping of genetic interactions for de novo fatty acid 
            synthesis identifies C12orf49 as a regulator of lipid metabolism.
  JOURNAL   Nat Metab. 2020 Jun;2(6):499-513. doi: 10.1038/s42255-020-0211-z. 
            Epub 2020 Jun 1.
  PUBMED    32694731
REFERENCE   2  (bases 1 to 14681)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s42255-020-0211-z"; journalName: "Nat Metab"; date: 
            "2020-06"; volume: "2"; issue: "6"; pages: "499-513"
COMMENT     Sequence Label: LCV2_mClover3
FEATURES             Location/Qualifiers
     source          1..14681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             3015..3809
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3069..3088)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3679..3698
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_RNA        3835..3910
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3972..4183
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4202..4211
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4208..8311
                     /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             4208..8311
                     /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             8312..8359
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8360..8383
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8393..8446
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(8453..8455,8456..9166)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     9103..9124
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             9176..9232
                     /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             9233..9829
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     9726..9746
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9845..10433
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9898..9918)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10316..10327)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10505..10738
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10764..10781)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10770..10994
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11040..11468
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11127..11146)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11337..11358
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11477..11497)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11521..11731
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        11543..11739
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      11590..11725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11652..11671
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11787..11834
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11853..12227
                     /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    12357..12478
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12394..12413)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12448..12467
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12527..12543)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12527..12543)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12540..12562)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12551..12567
                     /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(12575..12581,12582..12599,12600..12605))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12620..12641
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12758..12775)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12929..13517)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13009..13028)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13688..14479,14480..14548))
                     /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     14311..14330
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14549..14653)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccggga gacggcagca agcgaaccgg
     2881 aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg
     2941 ctttcttgcc gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat
     3001 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3061 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3121 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3181 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3241 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3301 aagtgccggg gcaggatctc ctgtcatccc accttgctcc tgccgagaaa gtatccatca
     3361 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3421 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3481 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3541 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3601 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3661 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3721 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3781 tctatcgcct tcttgacgag ttcttctgaa ttgaaaaagg aagagtacgt ctccgtttta
     3841 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     3901 gagtcggtgc ttttttgaat tcgctagcta ggtcttgaaa ggagtgggaa ttggctccgg
     3961 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     4021 cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     4081 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     4141 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc
     4201 tgccaccatg gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
     4261 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     4321 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     4381 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     4441 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     4501 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     4561 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     4621 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     4681 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     4741 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     4801 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     4861 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     4921 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     4981 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     5041 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     5101 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     5161 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     5221 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     5281 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     5341 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     5401 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     5461 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     5521 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     5581 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     5641 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt
     5701 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     5761 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     5821 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
     5881 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     5941 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     6001 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     6061 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     6121 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     6181 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     6241 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
     6301 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     6361 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     6421 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     6481 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     6541 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     6601 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     6661 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     6721 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac
     6781 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     6841 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
     6901 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     6961 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
     7021 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
     7081 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     7141 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     7201 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     7261 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     7321 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     7381 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
     7441 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
     7501 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
     7561 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
     7621 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     7681 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     7741 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     7801 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     7861 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     7921 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     7981 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
     8041 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
     8101 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     8161 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
     8221 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     8281 acggatcgac ctgtctcagc tgggaggcga caagcgacct gccgccacaa agaaggctgg
     8341 acaggctaag aagaagaaag attacaaaga cgatgacgat aagggcagtg gagagggcag
     8401 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccactcg aggtgagcaa
     8461 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     8521 cggccacaag ttcagcgtcc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac
     8581 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     8641 cttcggctac ggcgtggcct gcttcagccg ctaccccgac cacatgaagc agcacgactt
     8701 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatctctt tcaaggacga
     8761 cggtacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     8821 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     8881 caacttcaac agccactacg tctatatcac ggccgacaag cagaagaact gcatcaaggc
     8941 taacttcaag atccgccaca acgttgagga cggcagcgtg cagctcgccg accactacca
     9001 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcca
     9061 tcagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     9121 cgtgaccgcc gccgggatta cacatggcat ggacgagctg tacaagggat ccggcgcaac
     9181 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgaccgagta
     9241 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     9301 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     9361 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     9421 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     9481 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     9541 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     9601 cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     9661 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     9721 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     9781 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt
     9841 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     9901 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     9961 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
    10021 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
    10081 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
    10141 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
    10201 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
    10261 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
    10321 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
    10381 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
    10441 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
    10501 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
    10561 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
    10621 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
    10681 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagg
    10741 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
    10801 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
    10861 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
    10921 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
    10981 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
    11041 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
    11101 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
    11161 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
    11221 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
    11281 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
    11341 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
    11401 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
    11461 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
    11521 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
    11581 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
    11641 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
    11701 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
    11761 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca
    11821 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg
    11881 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
    11941 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
    12001 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
    12061 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
    12121 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
    12181 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag
    12241 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
    12301 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
    12361 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
    12421 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
    12481 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
    12541 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
    12601 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
    12661 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
    12721 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
    12781 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
    12841 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
    12901 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
    12961 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
    13021 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
    13081 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
    13141 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
    13201 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
    13261 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
    13321 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
    13381 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
    13441 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
    13501 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
    13561 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
    13621 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
    13681 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
    13741 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
    13801 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
    13861 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
    13921 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
    13981 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
    14041 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
    14101 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
    14161 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
    14221 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
    14281 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
    14341 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
    14401 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
    14461 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
    14521 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
    14581 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
    14641 taggggttcc gcgcacattt ccccgaaaag tgccacctga c
//
