LOCUS       sequence-307997-       13787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral plasmid expressing mCherry, Cas9 and a gRNA targeting 
            PSMD1 (core essential gene).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13787)
  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 13787)
  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_mCherry_PSMD1_sgRNA_1
FEATURES             Location/Qualifiers
     source          1..13787
                     /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"
     misc_RNA        2878..2953
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3015..3226
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3245..3254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3251..7354
                     /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             3251..7354
                     /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             7355..7402
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7403..7426
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7436..7489
                     /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             7496..8200
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(7638..7656)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7906..7925
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             8210..8266
                     /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             8267..8863
                     /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     8760..8780
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8879..9467
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8932..8952)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9350..9361)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9539..9772
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9798..9815)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9804..10028
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10074..10502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10161..10180)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10371..10392
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10511..10531)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10516..10845
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10696..10831
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10758..10777
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10893..10940
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10959..11333
                     /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    11463..11584
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11500..11519)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11554..11573
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11633..11649)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11633..11649)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11646..11668)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11657..11673
                     /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(11681..11687,11688..11705,11706..11711))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11726..11747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11864..11881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12035..12623)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12115..12134)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12794..13585,13586..13654))
                     /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     13417..13436
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13655..13759)
                     /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 aacaccgtgt gcgctacgga gctgcaagtt
     2881 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     2941 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
     3001 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     3061 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     3121 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     3181 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
     3241 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
     3301 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     3361 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     3421 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     3481 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     3541 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     3601 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     3661 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     3721 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     3781 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     3841 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     3901 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     3961 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     4021 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     4081 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     4141 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     4201 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     4261 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     4321 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     4381 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     4441 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     4501 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     4561 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     4621 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     4681 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     4741 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     4801 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     4861 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     4921 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     4981 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     5041 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     5101 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     5161 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     5221 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     5281 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     5341 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     5401 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     5461 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     5521 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     5581 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     5641 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     5701 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     5761 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     5821 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     5881 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     5941 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     6001 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     6061 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     6121 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     6181 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     6241 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     6301 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     6361 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     6421 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     6481 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     6541 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
     6601 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
     6661 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     6721 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     6781 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     6841 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     6901 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     6961 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     7021 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     7081 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     7141 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     7201 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
     7261 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     7321 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc
     7381 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggca gtggagaggg
     7441 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccac tcgaggtgag
     7501 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     7561 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     7621 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     7681 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     7741 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     7801 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     7861 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     7921 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     7981 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     8041 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     8101 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     8161 gggccgccac tccaccggcg gcatggacga gctgtacaag ggatccggcg caacaaactt
     8221 ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc
     8281 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     8341 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     8401 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     8461 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     8521 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     8581 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     8641 caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     8701 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     8761 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     8821 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
     8881 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     8941 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     9001 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     9061 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     9121 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     9181 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     9241 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     9301 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     9361 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     9421 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
     9481 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     9541 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     9601 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     9661 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     9721 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt
     9781 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     9841 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     9901 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     9961 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
    10021 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
    10081 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
    10141 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
    10201 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
    10261 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
    10321 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
    10381 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
    10441 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
    10501 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
    10561 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
    10621 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
    10681 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
    10741 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
    10801 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
    10861 tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata
    10921 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg
    10981 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
    11041 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
    11101 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
    11161 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
    11221 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
    11281 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc
    11341 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
    11401 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
    11461 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
    11521 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
    11581 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
    11641 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
    11701 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
    11761 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
    11821 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
    11881 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
    11941 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
    12001 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
    12061 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
    12121 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
    12181 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
    12241 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
    12301 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
    12361 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
    12421 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
    12481 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
    12541 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
    12601 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
    12661 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
    12721 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
    12781 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
    12841 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
    12901 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
    12961 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
    13021 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
    13081 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
    13141 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
    13201 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
    13261 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
    13321 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
    13381 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
    13441 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
    13501 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
    13561 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
    13621 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
    13681 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
    13741 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgac
//
