LOCUS Exported 9954 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral expression of SARS-CoV-2 protein; transient expression and generate lentivirus. ACCESSION . VERSION . KEYWORDS pLVX-EF1alpha-SARS-CoV-2-nsp15-2xStrep-IRES-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9954) AUTHORS Gordon DE, et al. TITLE A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug Repurposing JOURNAL BioRxiv March 2020 REFERENCE 2 (bases 1 to 9954) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9954 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 1..634 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 681..806 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1303..1536 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1721..1765 /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 2028..2143 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1 (lacking the first T)" promoter 2338..3519 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2568..3510 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3467..3487 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 4609..4632 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 4669..4692 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" misc_feature 4704..5277 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4871..4888) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5098..5117 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5310..5909 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(5310..5329) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5806..5826 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 5923..6511 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5976..5996) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6394..6405) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6718..7351 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" primer_bind complement(7479..7495) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7479..7495) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(7492..7514) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 7503..7519 /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(7527..7557) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7572..7593 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7710..7727) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7881..8469) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7961..7980) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8640..9500) /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 9263..9282 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(9501..9605) /gene="bla" /label=AmpR promoter polyA_signal 9653..9787 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(9690..9709) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 9744..9763 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2161 acagcagaga tccagtttat cgatgagtaa ttcatacaaa aggactcgcc cctgccttgg 2221 ggaatcccag ggaccgtcgt taaactccca ctaacgtaga acccagagat cgctgcgttc 2281 ccgccccctc acccgcccgc tctcgtcatc actgaggtgg agaagagcat gcgtgaggct 2341 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag 2401 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg 2461 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag 2521 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt 2581 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt 2641 ccacgcccct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 2701 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 2761 cttgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 2821 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 2881 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 2941 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 3001 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 3061 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 3121 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 3181 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 3241 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 3301 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 3361 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 3421 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 3481 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gatctatttc cggtgaattc 3541 gccgccacca tgagtcttga aaacgtcgcc ttcaacgtag taaataaggg ccactttgat 3601 ggtcaacagg gagaagtacc cgtgtctatc atcaataaca ccgtttacac gaaggttgac 3661 ggcgttgatg ttgaactgtt cgaaaataag acgacactcc cggtgaacgt agctttcgag 3721 ctttgggcga agcgaaatat aaaacctgtc cctgaagtta aaattctcaa taatcttggg 3781 gtagacatcg cggctaatac agtcatatgg gactataagc gggacgctcc ggcgcatata 3841 tctaccatcg gagtgtgctc aatgacagat attgccaaga aacctacaga gacgatttgc 3901 gctcctctta ccgtattttt tgacggcagg gtcgacggac aagtggatct gttccgaaac 3961 gcgcgcaatg gtgttctcat cactgagggt agtgtaaagg gactccagcc tagtgtcgga 4021 cctaagcaag cctctttgaa tggggtcact ctgatcggcg aggctgttaa gacgcaattt 4081 aactactata agaaggtcga tggcgtcgtg cagcagcttc ctgaaacata ctttacacag 4141 tctcgcaact tgcaagaatt taaaccgcgc tcacagatgg aaatagactt tctcgaactg 4201 gcaatggacg agttcataga gcggtacaaa ttggaagggt atgcctttga acacattgtt 4261 tatggtgact tttctcacag ccagttgggt ggtttgcatc tcctcatagg gctggccaaa 4321 aggttcaaag agagcccctt tgaattggag gacttcatac ctatggattc aacagttaag 4381 aattacttca ttacggatgc gcagacaggc agtagtaagt gtgtctgctc cgtaattgac 4441 ctcctcttgg acgactttgt agagataatt aagtcccagg atcttagtgt cgtatccaag 4501 gtcgttaagg tcacaattga ttacaccgag attagtttta tgctgtggtg taaagacggt 4561 cacgtcgaga ccttctatcc aaagttgcaa ctcgaaggcg gcgggggatg gagccatcca 4621 caattcgaga aaggcggtgg ttcaggagga ggtagcgggg gtggatcatg gtcacatccg 4681 cagtttgaaa agtaaggatc ccgcccctct ccctcccccc cccctaacgt tactggccga 4741 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg 4801 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg 4861 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt 4921 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac 4981 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca 5041 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg 5101 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg 5161 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa 5221 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataagct 5281 tgccacaacc cacaaggaga cgaccttcca tgaccgagta caagcccacg gtgcgcctcg 5341 ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc gccgactacc 5401 ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc gagctgcaag 5461 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg 5521 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga 5581 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag 5641 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct 5701 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg 5761 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct 5821 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct 5881 ggtgcatgac ccgcaagccc ggtgcctaga cgcgtctgga acaatcaacc tctggattac 5941 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga 6001 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc 6061 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa 6121 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc 6181 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc 6241 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 6301 gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg 6361 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct 6421 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg 6481 agtcggatct ccctttgggc cgcctccccg cctggaatta attctgcagt cgagacctag 6541 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc 6601 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac 6661 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag aggggactgg 6721 aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac 6781 aaggctactt ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga 6841 cctttggatg gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata 6901 aaggagagaa caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga 6961 gagaagtgtt agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc 7021 tgcatccgga gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg 7081 gggactttcc agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct 7141 gcatataagc agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc 7201 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga 7261 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga 7321 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag 7381 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggccttgaca ttgctagcgt 7441 ttaccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga 7501 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 7561 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 7621 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 7681 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 7741 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 7801 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 7861 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 7921 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 7981 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 8041 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 8101 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 8161 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 8221 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 8281 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 8341 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 8401 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 8461 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 8521 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 8581 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 8641 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 8701 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 8761 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 8821 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 8881 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 8941 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 9001 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 9061 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 9121 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 9181 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 9241 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 9301 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 9361 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 9421 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 9481 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 9541 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 9601 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcaacttgtt 9661 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 9721 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 9781 ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca ccccataccc 9841 tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt cctctgaatt 9901 attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag tagt //