LOCUS       sequence-166503-       15069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for the delivery of multiple sgRNAs targeting 
            different genes with constitutive Cas9 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15069)
  AUTHORS   Cao J, Wu L, Zhang SM, Lu M, Cheung WK, Cai W, Gale M, Xu Q, Yan Q
  TITLE     An easy and efficient inducible CRISPR/Cas9 platform with improved 
            specificity for multiple gene targeting.
  JOURNAL   Nucleic Acids Res. 2016 Nov 2;44(19):e149. Epub 2016 Jul 25.
  PUBMED    27458201
REFERENCE   2  (bases 1 to 15069)
  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: "Nucleic 
            Acids Res."; date: "2016-11-2"; volume: "44(19)"; pages: "e149. Epub
            2016 Jul 25"
COMMENT     Sequence Label: Lenti-multi-CRISPR
FEATURES             Location/Qualifiers
     source          1..15069
                     /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)"
     promoter        join(2880..2885,2886..2903,2904..2910)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2964..3623
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3030..3049)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3965..4270
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4149..4168
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        4309..4384
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4677..4925
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4934..5009
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5071..5282
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5301..5310
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5307..9410
                     /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             5307..9410
                     /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             9411..9458
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9459..9482
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9492..9548
                     /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             9549..10145
                     /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     10042..10062
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    10161..10749
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10214..10234)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10632..10643)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10821..11054
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(11080..11097)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11086..11310
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11356..11784
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11443..11462)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11653..11674
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11793..11813)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11798..12127
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11978..12113
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12040..12059
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12175..12222
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12241..12615
                     /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    12745..12866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12782..12801)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12836..12855
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12915..12931)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12915..12931)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12928..12950)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12939..12955
                     /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(12963..12969,12970..12987,12988..12993))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13008..13029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13146..13163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13317..13905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13397..13416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14076..14867,14868..14936))
                     /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     14699..14718
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14937..15041)
                     /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 acgattaggc accccaggct
     2881 ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat ccggcgagat
     2941 tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata
     3001 tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct
     3061 ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc
     3121 acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
     3181 tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca
     3241 ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt
     3301 tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct
     3361 atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt
     3421 tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
     3481 tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc
     3541 atgccgtctg tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg
     3601 atgagtggca gggcggggcg taaagatctg gatccggctt actaaaagcc agataacagt
     3661 atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa
     3721 gtatgtcaaa aagaggtgtg ctatgaagca gcgtattaca gtgacagttg acagcgacag
     3781 ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg
     3841 cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg
     3901 gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa cagggactgg
     3961 tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     4021 tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc
     4081 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     4141 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga
     4201 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     4261 gggaatataa atgtcaggct cccttataca cagccagtct gcagctgggt tttagagcta
     4321 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     4381 gtgctttttt aagcttggcg taactagatc ttgagacaaa tggcagtatt catccacaat
     4441 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagagtccg gcaagtttgt
     4501 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     4561 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     4621 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccagagagg
     4681 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     4741 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     4801 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     4861 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     4921 acaccgcgtc tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     4981 aacttgaaaa agtggcaccg agtcggtgct tttttgaatt cgctagctag gtcttgaaag
     5041 gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     5101 agaagttggg gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa
     5161 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     5221 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     5281 aggaccggtt ctagagcgct gccaccatgg acaagaagta cagcatcggc ctggacatcg
     5341 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     5401 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     5461 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     5521 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     5581 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     5641 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     5701 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     5761 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     5821 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     5881 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     5941 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     6001 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     6061 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     6121 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     6181 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     6241 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     6301 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     6361 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     6421 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     6481 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     6541 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     6601 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     6661 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     6721 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     6781 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     6841 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     6901 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     6961 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     7021 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     7081 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     7141 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     7201 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     7261 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     7321 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     7381 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     7441 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     7501 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     7561 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     7621 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     7681 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     7741 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     7801 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     7861 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     7921 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     7981 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     8041 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     8101 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     8161 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     8221 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     8281 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     8341 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     8401 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     8461 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     8521 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     8581 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     8641 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     8701 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     8761 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     8821 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     8881 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     8941 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     9001 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     9061 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     9121 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     9181 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     9241 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     9301 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     9361 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgacctg
     9421 ccgccacaaa gaaggctgga caggctaaga agaagaaaga ttacaaagac gatgacgata
     9481 agggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc
     9541 ctggaccgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     9601 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     9661 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     9721 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     9781 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     9841 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     9901 aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc
     9961 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
    10021 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
    10081 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
    10141 cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
    10201 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
    10261 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
    10321 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
    10381 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
    10441 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
    10501 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
    10561 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
    10621 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
    10681 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
    10741 cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
    10801 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
    10861 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
    10921 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
    10981 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
    11041 gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
    11101 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
    11161 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
    11221 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
    11281 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
    11341 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
    11401 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
    11461 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
    11521 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
    11581 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
    11641 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
    11701 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
    11761 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
    11821 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
    11881 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
    11941 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
    12001 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
    12061 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
    12121 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
    12181 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
    12241 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
    12301 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
    12361 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
    12421 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
    12481 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
    12541 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
    12601 gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg
    12661 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
    12721 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
    12781 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
    12841 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
    12901 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
    12961 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
    13021 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
    13081 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
    13141 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
    13201 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
    13261 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
    13321 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
    13381 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
    13441 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
    13501 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
    13561 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
    13621 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
    13681 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
    13741 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
    13801 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
    13861 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
    13921 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
    13981 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
    14041 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
    14101 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
    14161 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
    14221 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
    14281 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
    14341 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
    14401 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
    14461 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
    14521 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
    14581 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
    14641 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
    14701 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
    14761 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
    14821 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
    14881 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
    14941 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
    15001 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
    15061 ccacctgac
//
