LOCUS Exported 9276 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-orf8-2xStrep-IRES-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9276) 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 9276) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9276 /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 3931..3954 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 3991..4014 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" misc_feature 4026..4599 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4193..4210) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4420..4439 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 4632..5231 /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(4632..4651) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5128..5148 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 5245..5833 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5298..5318) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5716..5727) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6040..6673 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" primer_bind complement(6801..6817) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6801..6817) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6814..6836) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6825..6841 /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(6849..6879) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6894..6915 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7032..7049) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7203..7791) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7283..7302) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7962..8822) /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 8585..8604 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8823..8927) /gene="bla" /label=AmpR promoter polyA_signal 8975..9109 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(9012..9031) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 9066..9085 /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 tgaagtttct cgtgttcttg ggcattatca ccacagtcgc ggcttttcat 3601 caagaatgct ctttgcagtc ttgtacacag catcaacctt atgtcgtcga tgatccttgc 3661 ccgattcact tttacagtaa atggtatatc agggtagggg cccgaaaaag cgcaccattg 3721 attgaattgt gtgtggacga ggcagggagc aaaagtccga ttcaatacat tgacatcggt 3781 aattatactg tcagttgttc cccctttacg atcaactgcc aagaacccaa actggggagc 3841 ttggtggtcc gatgttcctt ctatgaagat tttcttgagt atcacgatgt cagggtggtc 3901 cttgacttta tactcgaagg cggcggggga tggagccatc cacaattcga gaaaggcggt 3961 ggttcaggag gaggtagcgg gggtggatca tggtcacatc cgcagtttga aaagtaagga 4021 tcccgcccct ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc 4081 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg 4141 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc 4201 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga 4261 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg 4321 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag 4381 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc 4441 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct 4501 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac 4561 cacggggacg tggttttcct ttgaaaaaca cgatgataag cttgccacaa cccacaagga 4621 gacgaccttc catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc 4681 cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg 4741 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg 4801 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga 4861 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg 4921 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc 4981 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca 5041 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc 5101 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca 5161 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc 5221 ccggtgccta gacgcgtctg gaacaatcaa cctctggatt acaaaatttg tgaaagattg 5281 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct 5341 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg 5401 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact 5461 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc 5521 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc 5581 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag 5641 ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc 5701 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg 5761 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg 5821 gccgcctccc cgcctggaat taattctgca gtcgagacct agaaaaacat ggagcaatca 5881 caagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg 5941 aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag 6001 ctgtagatct tagccacttt ttaaaagaaa agaggggact ggaagggcta attcactccc 6061 aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt 6121 agcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga tggtgctaca 6181 agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag aacaccagct 6241 tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagtg ttagagtgga 6301 ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg gagtacttca 6361 agaactgctg atatcgagct tgctacaagg gactttccgc tggggacttt ccagggaggc 6421 gtggcctggg cgggactggg gagtggcgag ccctcagatc ctgcatataa gcagctgctt 6481 tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa 6541 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt 6601 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg 6661 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag 6721 aaatgaatat cagagagtga gaggccttga cattgctagc gtttaccgtc gacctctagc 6781 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 6841 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 6901 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 6961 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 7021 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 7081 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 7141 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 7201 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 7261 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 7321 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 7381 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 7441 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 7501 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 7561 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 7621 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 7681 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 7741 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 7801 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 7861 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 7921 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 7981 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 8041 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 8101 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 8161 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 8221 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 8281 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 8341 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 8401 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 8461 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 8521 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 8581 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 8641 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 8701 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 8761 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 8821 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 8881 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 8941 aaagtgccac ctgacgtcga cggatcggga gatcaacttg tttattgcag cttataatgg 9001 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 9061 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatca actggataac 9121 tcaagctaac caaaatcatc ccaaacttcc caccccatac cctattacca ctgccaatta 9181 cctgtggttt catttactct aaacctgtga ttcctctgaa ttattttcat tttaaagaaa 9241 ttgtatttgt taaatatgta ctacaaactt agtagt //