LOCUS       sequence-308002-       14753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral backbone for cloning and expression of U6 driven gRNAs 
            with Esp3I/BsmBI cloning sites, puromycin selection and BFP2 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14753)
  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 14753)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s42255-020-0211-z"; journalName: "Nat Metab"; date: 
            "2020-06"; volume: "2"; issue: "6"; pages: "499-513"
COMMENT     Sequence Label: LCV2_BFP2
FEATURES             Location/Qualifiers
     source          1..14753
                     /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="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIKV
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLLE
                     FRTAAGITLGMDELYK"
     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        11482..11811
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11662..11797
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11724..11743
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11859..11906
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11925..12299
                     /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    12429..12550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12466..12485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12520..12539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12599..12615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12599..12615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12612..12634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12623..12639
                     /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(12647..12653,12654..12671,12672..12677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12692..12713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12830..12847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13001..13589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13081..13100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13760..14551,14552..14620))
                     /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     14383..14402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14621..14725)
                     /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 ttcagcgtga ggggcgaggg cgagggcgat gccaccaacg gcaagctgac
     8581 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     8641 cctgagccac ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     8701 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     8761 cggcacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctag tgaaccgcat
     8821 cgagctgaag ggcgtcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     8881 caacttcaac agccacaaca tctatatcat ggccgtcaag cagaagaacg gcatcaaggt
     8941 gaacttcaag atccgccaca acgtggagga cggcagcgtg cagctcgccg accactacca
     9001 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacagccact acctgagcac
     9061 ccagtccgtg ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     9121 ccgcaccgcc gccgggatca ctctcggcat 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 caaccaggtg tggaaagtcc
    11581 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
    11641 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
    11701 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
    11761 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
    11821 ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat
    11881 agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca
    11941 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
    12001 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
    12061 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
    12121 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
    12181 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
    12241 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac
    12301 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
    12361 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
    12421 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
    12481 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
    12541 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
    12601 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
    12661 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
    12721 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
    12781 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
    12841 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
    12901 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
    12961 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
    13021 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
    13081 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
    13141 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
    13201 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
    13261 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
    13321 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
    13381 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
    13441 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
    13501 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
    13561 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
    13621 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
    13681 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
    13741 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
    13801 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
    13861 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
    13921 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
    13981 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
    14041 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
    14101 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
    14161 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
    14221 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
    14281 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
    14341 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
    14401 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
    14461 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
    14521 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
    14581 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
    14641 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
    14701 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gac
//
