LOCUS       sequence-360098-       13479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid with Cas9, two U6 promoters and two gRNA scaffolds that 
            allows for inserting two gRNAs for combinatorial CRISPR Screen or 
            double-knock-out (DKO) screen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13479)
  AUTHORS   Tang S, Wu X, Liu J, Zhang Q, Wang X, Shao S, Gokbag B, Fan K, Liu 
            X, Li F, Cheng L, Li L
  TITLE     Generation of dual-gRNA library for combinatorial CRISPR screening 
            of synthetic lethal gene pairs.
  JOURNAL   STAR Protoc. 2022 Aug 24;3(3):101556. doi: 
            10.1016/j.xpro.2022.101556. eCollection 2022 Sep 16.
  PUBMED    36060092
REFERENCE   2  (bases 1 to 13479)
  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: "STAR 
            Protoc."; date: "2022-08-24"; volume: "3(3):101556. doi"; pages: " 
            10.1016/j.xpro.2022.101556. eCollection 2022 Sep 16"
COMMENT     Sequence Label: lentiCRISPR_DKO
FEATURES             Location/Qualifiers
     source          1..13479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..234
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     189..209
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             252..432
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    479..604
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1097..1330
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1515..1559
                     /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    1857..1974
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2025..2265
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     2025..2045
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2196..2215
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2409..2722
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2641..2661
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        2761..2836
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2898..3109
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3128..3137
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3134..7237
                     /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             3134..7237
                     /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             7238..7285
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7286..7309
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7319..7375
                     /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             7376..7972
                     /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     7869..7889
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7988..8576
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8041..8061)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8459..8470)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8648..8881
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8907..8924)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8913..9137
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9183..9611
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9270..9289)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9480..9501
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9620..9640)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9625..9954
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9805..9940
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9867..9886
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10002..10049
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10068..10442
                     /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    10572..10693
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10609..10628)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10663..10682
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10742..10758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10742..10758)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10755..10777)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10766..10782
                     /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(10790..10796,10797..10814,10815..10820))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10835..10856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10973..10990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11144..11732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11224..11243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11903..12694,12695..12763))
                     /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     12526..12545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12764..12868)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12943..12962)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(13134..13479,1..34)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
       61 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      121 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      181 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt
      241 ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
      301 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
      361 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
      421 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
      481 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
      541 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
      601 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
      661 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
      721 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
      781 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
      841 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
      901 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac
      961 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     1021 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     1081 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     1141 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     1201 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     1261 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     1321 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     1381 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     1441 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     1501 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     1561 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     1621 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     1681 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc
     1741 ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga
     1801 acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt
     1861 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     1921 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     1981 tattacaggg acagcagaga tccagtttgg ttaattaagg taccgagggc ctatttccca
     2041 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
     2101 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     2161 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     2221 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggactctag aaccaggatg
     2281 ggcaccaccc gtttaagagc taagctggaa acagcatagc aagtttaaat aaggctagtc
     2341 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgtactga gtcgcccagt
     2401 ctcagataga tccgacgccg ccatctctag gcccgcgccg gccccctcgc acagacttgt
     2461 gggagaagct cggctactcc cctgccccgg ttaatttgca tataatattt cctagtaact
     2521 atagaggctt aatgtgcgat aaaagacaga taatctgttc tttttaatac tagctacatt
     2581 ttacatgata ggcttggatt tctataagag atacaaatac taaattatta ttttaaaaaa
     2641 cagcacaaaa ggaaactcac cctaactgta aagtaattgt gtgttttgag actataaata
     2701 tcccttggag aaaagccttg ttcaccggag acggcttctt gctctttttt gtacgtctct
     2761 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     2821 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg
     2881 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     2941 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     3001 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     3061 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta
     3121 gagcgctgcc accatggaca agaagtacag catcggcctg gacatcggca ccaactctgt
     3181 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
     3241 caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg
     3301 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
     3361 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
     3421 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
     3481 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
     3541 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
     3601 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
     3661 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
     3721 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
     3781 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
     3841 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
     3901 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
     3961 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
     4021 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
     4081 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
     4141 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
     4201 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
     4261 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
     4321 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
     4381 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
     4441 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
     4501 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
     4561 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac
     4621 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
     4681 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
     4741 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
     4801 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
     4861 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
     4921 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
     4981 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
     5041 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
     5101 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
     5161 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
     5221 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
     5281 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
     5341 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
     5401 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
     5461 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
     5521 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
     5581 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
     5641 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt
     5701 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
     5761 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
     5821 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
     5881 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
     5941 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
     6001 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
     6061 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
     6121 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
     6181 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
     6241 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
     6301 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
     6361 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
     6421 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
     6481 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
     6541 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
     6601 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
     6661 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
     6721 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
     6781 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
     6841 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
     6901 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
     6961 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
     7021 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
     7081 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
     7141 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
     7201 cgagacacgg atcgacctgt ctcagctggg aggcgacaag cgacctgccg ccacaaagaa
     7261 ggctggacag gctaagaaga agaaagatta caaagacgat gacgataagg gatccggcgc
     7321 aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgaccga
     7381 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     7441 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     7501 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     7561 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     7621 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     7681 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     7741 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     7801 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     7861 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     7921 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
     7981 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     8041 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     8101 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     8161 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     8221 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     8281 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     8341 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     8401 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     8461 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     8521 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     8581 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     8641 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     8701 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     8761 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     8821 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     8881 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     8941 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     9001 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     9061 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     9121 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     9181 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     9241 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     9301 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     9361 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     9421 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     9481 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     9541 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     9601 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     9661 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     9721 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     9781 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     9841 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     9901 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     9961 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
    10021 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
    10081 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
    10141 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
    10201 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
    10261 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
    10321 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
    10381 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
    10441 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
    10501 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
    10561 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
    10621 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
    10681 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
    10741 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
    10801 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
    10861 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
    10921 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
    10981 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
    11041 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
    11101 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
    11161 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
    11221 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
    11281 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
    11341 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
    11401 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
    11461 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
    11521 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
    11581 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
    11641 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
    11701 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
    11761 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
    11821 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
    11881 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
    11941 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
    12001 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
    12061 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
    12121 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
    12181 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
    12241 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
    12301 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
    12361 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
    12421 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
    12481 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
    12541 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
    12601 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
    12661 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
    12721 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    12781 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    12841 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
    12901 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
    12961 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
    13021 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
    13081 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
    13141 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
    13201 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
    13261 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
    13321 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
    13381 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
    13441 tgcccagtac atgaccttat gggactttcc tacttggca
//
