LOCUS       sequence-307983-       14747 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 EGFP 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14747)
  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 14747)
  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_EGFP
FEATURES             Location/Qualifiers
     source          1..14747
                     /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(8447..8449,8450..9160)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(8447..8449,8450..9160)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(8750..8769)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9097..9118
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             9170..9226
                     /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             9227..9823
                     /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     9720..9740
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9839..10427
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9892..9912)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10310..10321)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10499..10732
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(10758..10775)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10764..10988
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11034..11462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11121..11140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11331..11352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11471..11491)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11476..11805
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11656..11791
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11718..11737
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11853..11900
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11919..12293
                     /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    12423..12544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12460..12479)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12514..12533
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12593..12609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12593..12609)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12606..12628)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12617..12633
                     /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(12641..12647,12648..12665,12666..12671))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12686..12707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12824..12841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12995..13583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13075..13094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13754..14545,14546..14614))
                     /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     14377..14396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14615..14719)
                     /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 ggcccagtga gcaagggcga
     8461 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     8521 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     8581 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     8641 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     8701 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     8761 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     8821 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     8881 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     8941 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     9001 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     9061 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     9121 cgccgccggg atcactctcg gcatggacga gctgtacaag ggatccggcg caacaaactt
     9181 ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc
     9241 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     9301 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     9361 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     9421 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     9481 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     9541 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     9601 caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     9661 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     9721 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     9781 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
     9841 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     9901 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     9961 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
    10021 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
    10081 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
    10141 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
    10201 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
    10261 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
    10321 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
    10381 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
    10441 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
    10501 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
    10561 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
    10621 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
    10681 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt
    10741 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
    10801 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
    10861 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
    10921 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
    10981 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
    11041 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
    11101 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
    11161 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
    11221 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
    11281 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
    11341 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
    11401 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
    11461 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
    11521 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
    11581 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
    11641 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
    11701 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
    11761 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
    11821 tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata
    11881 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg
    11941 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
    12001 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
    12061 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
    12121 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
    12181 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
    12241 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc
    12301 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
    12361 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
    12421 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
    12481 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
    12541 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
    12601 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
    12661 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
    12721 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
    12781 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
    12841 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
    12901 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
    12961 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
    13021 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
    13081 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
    13141 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
    13201 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
    13261 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
    13321 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
    13381 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
    13441 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
    13501 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
    13561 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
    13621 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
    13681 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
    13741 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
    13801 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
    13861 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
    13921 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
    13981 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
    14041 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
    14101 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
    14161 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
    14221 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
    14281 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
    14341 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
    14401 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
    14461 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
    14521 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
    14581 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
    14641 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
    14701 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgac
//
