LOCUS       sequence-275159-       15635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lenti Crispr/Cas9 with mOrange fluorescent marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15635)
  AUTHORS   Jin J, Xu Y, Huo L, Ma L, Scott AW, Pizzi MP, Li Y, Wang Y, Yao X, 
            Song S, Ajani JA
  TITLE     An improved strategy for CRISPR/Cas9 gene knockout and subsequent 
            wildtype and mutant gene rescue.
  JOURNAL   PLoS One. 2020 Feb 13;15(2):e0228910. doi: 
            10.1371/journal.pone.0228910. eCollection 2020.
  PUBMED    32053639
REFERENCE   2  (bases 1 to 15635)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2020-02-13"; volume: "15(2):e0228910. doi"; pages: " 
            10.1371/journal.pone.0228910. eCollection 2020"
COMMENT     Sequence Label: pLentiCrispr-V2-mOrange (V2mO)
FEATURES             Location/Qualifiers
     source          1..15635
                     /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        4738..4813
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4875..5086
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5105..5114
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5111..9214
                     /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             5111..9214
                     /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             9215..9262
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9263..9286
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9296..9352
                     /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             9353..10060
                     /codon_start=1
                     /product="orange monomeric derivative of DsRed fluorescent 
                     protein (Shanet et al., 2004)"
                     /label=mOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     9766..9785
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             10061..10114
                     /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             10115..10711
                     /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     10608..10628
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    10727..11315
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10780..10800)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11198..11209)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11387..11620
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(11646..11663)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11652..11876
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11922..12350
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12009..12028)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12219..12240
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12359..12379)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12364..12693
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      12544..12679
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12606..12625
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12741..12788
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12807..13181
                     /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    13311..13432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13348..13367)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13402..13421
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13481..13497)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13481..13497)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13494..13516)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13505..13521
                     /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(13529..13535,13536..13553,13554..13559))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13574..13595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13712..13729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13883..14471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13963..13982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14642..15433,15434..15502))
                     /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     15265..15284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15503..15607)
                     /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 aacaccggag acggttgtaa atgagcacac
     2881 aaaatacaca tgctaaaata ttatattcta tgacctttat aaaatcaacc aaaatcttct
     2941 ttttaataac tttagtatca ataattagaa tttttatgtt cctttttgca aacttttaat
     3001 aaaaatgagc aaaataaaaa aacgctagtt ttagtaactc gcgttgtttt cttcaccttt
     3061 aataatagct actccaccac ttgttcctaa gcggtcagct cctgcttcaa tcattttttg
     3121 agcatcttca aatgttctaa ctccaccagc tgctttaact aaagcattgt ctttaacaac
     3181 tgacttcatt agtttaacat cttcaaatgt tgcacctgat tttgaaaatc ctgttgatgt
     3241 tttaacaaat tctaatccag cttcaacagc tatttcacaa gctttcatga tttcttcttt
     3301 tgttaataaa caattttcca taatacattt aacaacatgt gatccagctg ctttttttac
     3361 agctttcatg tcttctaaaa ctaattcata atttttgtct tttaatgcac caatatttaa
     3421 taccatatca atttctgttg caccatcttt aattgcttca gaaacttcga atgcttttgt
     3481 agctgttgtg catgcaccta gaggaaaacc tacaacattt gttattccta catttgtgcc
     3541 ttttaataat tctttacaat agcttgttca atatgaatta acacaaactg ttgcaaaatc
     3601 aaattcaatt gcttcatcac ataattgttt aatttcagct ttcgtagcat cttgttttaa
     3661 taatgtgtga tctatatatt tgtttagttt cattttttct cctatatatt catttttaat
     3721 tttaattctt taataatttc gtctacttta actttagcgt tttgaacaga ttcaccaaca
     3781 cctataaaat aaatttttag tttaggttca gttccacttg ggcgaacagc aaatcatgac
     3841 ttatcttcta aataaaattt tagtaagtct tgtcctggca tattatacat tccatcgatg
     3901 tagtcttcaa cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat
     3961 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
     4021 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
     4081 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
     4141 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
     4201 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
     4261 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
     4321 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
     4381 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
     4441 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
     4501 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga
     4561 tttggattta caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata
     4621 acgttatatc ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact
     4681 gcgctaaaaa taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt
     4741 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     4801 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
     4861 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     4921 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     4981 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     5041 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
     5101 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
     5161 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     5221 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     5281 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     5341 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     5401 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     5461 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     5521 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     5581 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     5641 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     5701 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     5761 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     5821 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     5881 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     5941 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     6001 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     6061 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     6121 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     6181 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     6241 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     6301 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     6361 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     6421 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     6481 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     6541 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     6601 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     6661 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     6721 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     6781 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     6841 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     6901 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     6961 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     7021 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     7081 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     7141 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     7201 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     7261 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     7321 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     7381 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     7441 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     7501 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     7561 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     7621 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     7681 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     7741 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     7801 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     7861 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     7921 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     7981 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     8041 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     8101 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     8161 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     8221 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     8281 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     8341 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     8401 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
     8461 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
     8521 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     8581 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     8641 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     8701 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     8761 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     8821 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     8881 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     8941 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     9001 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     9061 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
     9121 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     9181 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc
     9241 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat ccggcgcaac
     9301 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgatggtgag
     9361 caagggcgag gagaataaca tggccatcat caaggagttc atgcgcttca aggtgcgcat
     9421 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     9481 cgagggcttt cagaccgcta agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     9541 ggacatcctg tcccctcagt tcacctacgg ctccaaggcc tacgtgaagc accccgccga
     9601 catccccgac tacttcaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     9661 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     9721 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     9781 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     9841 gggcgagatc aagatgaggc tgaagctgaa ggacggcggc cactacacct ccgaggtcaa
     9901 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacatcg tcggcatcaa
     9961 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
    10021 gggccgccac tccaccggcg gcatggacga gctgtacaag gagggcagag gaagtcttct
    10081 aacatgcggt gacgtggagg agaatcccgg ccctaccgag tacaagccca cggtgcgcct
    10141 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
    10201 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
    10261 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
    10321 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
    10381 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
    10441 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
    10501 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
    10561 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
    10621 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
    10681 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga
    10741 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
    10801 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
    10861 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
    10921 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
    10981 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
    11041 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
    11101 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
    11161 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
    11221 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
    11281 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
    11341 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
    11401 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
    11461 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
    11521 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
    11581 tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg
    11641 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
    11701 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
    11761 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
    11821 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
    11881 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
    11941 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
    12001 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
    12061 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
    12121 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
    12181 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
    12241 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
    12301 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
    12361 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
    12421 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
    12481 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
    12541 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
    12601 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
    12661 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
    12721 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat
    12781 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca
    12841 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
    12901 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
    12961 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
    13021 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
    13081 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
    13141 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
    13201 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
    13261 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
    13321 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
    13381 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
    13441 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
    13501 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
    13561 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
    13621 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
    13681 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
    13741 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
    13801 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
    13861 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
    13921 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
    13981 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
    14041 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
    14101 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
    14161 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
    14221 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
    14281 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
    14341 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
    14401 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
    14461 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
    14521 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
    14581 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
    14641 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
    14701 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
    14761 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
    14821 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
    14881 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
    14941 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
    15001 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
    15061 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
    15121 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
    15181 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
    15241 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
    15301 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
    15361 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
    15421 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
    15481 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
    15541 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
    15601 ttccgcgcac atttccccga aaagtgccac ctgac
//
