LOCUS Exported 9834 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-nsp5-2xStrep-IRES-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9834) 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 9834) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9834 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..1182 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 231..1173 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1130..1150 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 2152..2175 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 2212..2235 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" misc_feature 2247..2820 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2414..2431) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2641..2660 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2853..3452 /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(2853..2872) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3349..3369 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 3466..4054 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3519..3539) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3937..3948) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4261..4894 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" primer_bind complement(5022..5038) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5022..5038) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5035..5057) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5046..5062 /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(5070..5100) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5115..5136 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5253..5270) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5424..6012) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5504..5523) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6183..7043) /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 6806..6825 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7044..7148) /gene="bla" /label=AmpR promoter polyA_signal 7196..7330 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7233..7252) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 7287..7306 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" LTR 7498..8131 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 8178..8303 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8800..9033 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 9218..9262 /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 9525..9640 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1 (lacking the first T)" ORIGIN 1 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 61 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 121 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 181 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 241 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 301 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg 361 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc 421 tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct 481 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc 541 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg 601 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg 661 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg 721 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca 781 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg 841 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt 901 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc 961 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 1021 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg 1081 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag 1141 acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggatctat ttccggtgaa 1201 ttcgccgcca ccatgtctgg ttttaggaaa atggcgttcc ccagcggtaa agttgaagga 1261 tgtatggtcc aagtaacctg tggtaccact acccttaatg ggctttggtt ggacgacgta 1321 gtctactgcc cccgacacgt aatctgcact agtgaggata tgcttaatcc caattacgaa 1381 gaccttttga ttcggaaatc caatcacaac ttcctggtcc aagcgggcaa cgtccaactc 1441 agggttattg gacatagtat gcagaattgc gtactgaagc tcaaagtcga tactgcaaac 1501 cccaagacgc ccaagtataa attcgtccga atccaaccag gccaaacatt ttccgtattg 1561 gcttgctata atggaagccc cagcggtgtc taccaatgtg caatgagacc aaactttacg 1621 ataaagggtt catttctgaa cggctcttgc ggttccgttg gttttaacat cgactatgac 1681 tgtgtatcct tttgctacat gcaccatatg gaactcccta ccggtgtcca cgccggtaca 1741 gatctggaag gaaatttcta cggtccgttc gttgaccggc aaaccgcgca agcggctgga 1801 accgacacaa cgattacagt gaatgtgctc gcgtggctgt acgcagcagt cataaacgga 1861 gacaggtggt ttctgaaccg atttacgacg actctcaatg acttcaacct tgttgcgatg 1921 aagtacaatt acgagccact cacccaggac catgttgata tcctgggtcc cctcagtgcc 1981 cagacaggga tcgcagttct cgatatgtgc gcgtcactga aggagcttct ccaaaatgga 2041 atgaatgggc ggaccatact tggttccgca ctcctcgaag atgaatttac tccatttgac 2101 gtggtcagac aatgcagtgg ggtcactttc cagctcgaag gcggcggggg atggagccat 2161 ccacaattcg agaaaggcgg tggttcagga ggaggtagcg ggggtggatc atggtcacat 2221 ccgcagtttg aaaagtaagg atcccgcccc tctccctccc ccccccctaa cgttactggc 2281 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg 2341 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct 2401 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca 2461 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg 2521 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct 2581 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa 2641 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt 2701 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa 2761 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 2821 gcttgccaca acccacaagg agacgacctt ccatgaccga gtacaagccc acggtgcgcc 2881 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact 2941 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc 3001 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg 3061 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg 3121 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg 3181 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg 3241 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg 3301 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct 3361 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca 3421 cctggtgcat gacccgcaag cccggtgcct agacgcgtct ggaacaatca acctctggat 3481 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 3541 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 3601 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 3661 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 3721 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 3781 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 3841 tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc 3901 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 3961 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 4021 acgagtcgga tctccctttg ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc 4081 tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct 4141 ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa 4201 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aagaggggac 4261 tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 4321 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 4381 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 4441 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 4501 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 4561 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 4621 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 4681 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 4741 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 4801 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 4861 agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt 4921 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggccttg acattgctag 4981 cgtttaccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg 5041 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 5101 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 5161 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 5221 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 5281 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 5341 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 5401 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 5461 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 5521 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 5581 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 5641 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 5701 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 5761 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 5821 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 5881 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 5941 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 6001 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 6061 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 6121 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 6181 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 6241 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 6301 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 6361 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 6421 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 6481 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 6541 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 6601 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 6661 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 6721 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 6781 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 6841 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 6901 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 6961 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 7021 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 7081 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 7141 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcaactt 7201 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 7261 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 7321 tgtctggatc aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata 7381 ccctattacc actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga 7441 attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagttgg 7501 aagggctaat tcactcccaa agaagacaag atatccttga tctgtggatc taccacacac 7561 aaggctactt ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga 7621 cctttggatg gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata 7681 aaggagagaa caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga 7741 gagaagtgtt agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc 7801 tgcatccgga gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg 7861 gggactttcc agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct 7921 gcatataagc agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc 7981 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga 8041 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga 8101 cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga 8161 aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa 8221 gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg 8281 agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa 8341 aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa 8401 gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta 8461 gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat 8521 tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca 8581 aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag 8641 cggccggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga 8701 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa 8761 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt 8821 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag 8881 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca 8941 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc 9001 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact 9061 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat 9121 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat 9181 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga 9241 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat 9301 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact 9361 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc 9421 aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag 9481 agacagatcc attcgattag tgaacggatc tcgacggtat cgcctttaaa agaaaagggg 9541 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa 9601 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca 9661 gcagagatcc agtttatcga tgagtaattc atacaaaagg actcgcccct gccttgggga 9721 atcccaggga ccgtcgttaa actcccacta acgtagaacc cagagatcgc tgcgttcccg 9781 ccccctcacc cgcccgctct cgtcatcact gaggtggaga agagcatgcg tgag //