LOCUS Exported 9096 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-orf6-2xStrep-IRES-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9096) 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 9096) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9096 /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 1414..1437 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 1474..1497 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" misc_feature 1509..2082 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(1676..1693) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 1903..1922 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2115..2714 /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(2115..2134) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2611..2631 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 2728..3316 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2781..2801) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3199..3210) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3523..4156 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" primer_bind complement(4284..4300) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4284..4300) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4297..4319) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4308..4324 /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(4332..4362) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4377..4398 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4515..4532) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4686..5274) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4766..4785) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5445..6305) /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 6068..6087 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6306..6410) /gene="bla" /label=AmpR promoter polyA_signal 6458..6592 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6495..6514) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6549..6568 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" LTR 6760..7393 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 7440..7565 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8062..8295 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8480..8524 /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 8787..8902 /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 ccatgtttca tcttgtcgat tttcaagtta cgattgcgga gatactcctc 1261 ataatcatgc ggacatttaa ggtttccatt tggaaccttg attacattat taacctgatt 1321 atcaagaatc tctccaagag cctgaccgag aataagtatt ctcagttgga cgaggagcag 1381 cctatggaaa tagacctcga aggcggcggg ggatggagcc atccacaatt cgagaaaggc 1441 ggtggttcag gaggaggtag cgggggtgga tcatggtcac atccgcagtt tgaaaagtaa 1501 ggatcccgcc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa 1561 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg 1621 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc 1681 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct 1741 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac 1801 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc 1861 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta 1921 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg 1981 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg 2041 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aagcttgcca caacccacaa 2101 ggagacgacc ttccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg 2161 tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca 2221 ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc 2281 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct 2341 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg 2401 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc 2461 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg 2521 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg 2581 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct 2641 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca 2701 agcccggtgc ctagacgcgt ctggaacaat caacctctgg attacaaaat ttgtgaaaga 2761 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 2821 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 2881 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 2941 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 3001 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 3061 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 3121 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 3181 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 3241 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 3301 tgggccgcct ccccgcctgg aattaattct gcagtcgaga cctagaaaaa catggagcaa 3361 tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg 3421 aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg 3481 cagctgtaga tcttagccac tttttaaaag aaaagagggg actggaaggg ctaattcact 3541 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg 3601 attagcagaa ctacacacca gggccagggg tcagatatcc actgaccttt ggatggtgct 3661 acaagctagt accagttgag ccagataagg tagaagaggc caataaagga gagaacacca 3721 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtgttagagt 3781 ggaggtttga cagccgccta gcatttcatc acgtggcccg agagctgcat ccggagtact 3841 tcaagaactg ctgatatcga gcttgctaca agggactttc cgctggggac tttccaggga 3901 ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg 3961 ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc 4021 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 4081 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 4141 tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca 4201 aagaaatgaa tatcagagag tgagaggcct tgacattgct agcgtttacc gtcgacctct 4261 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 4321 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4381 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4441 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4501 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4561 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4621 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4681 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4741 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4801 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4861 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4921 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4981 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 5041 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 5101 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 5161 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 5221 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 5281 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 5341 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5401 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 5461 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 5521 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 5581 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 5641 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 5701 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 5761 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 5821 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 5881 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 5941 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 6001 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 6061 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 6121 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 6181 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 6241 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 6301 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 6361 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6421 cgaaaagtgc cacctgacgt cgacggatcg ggagatcaac ttgtttattg cagcttataa 6481 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 6541 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaactggat 6601 aactcaagct aaccaaaatc atcccaaact tcccacccca taccctatta ccactgccaa 6661 ttacctgtgg tttcatttac tctaaacctg tgattcctct gaattatttt cattttaaag 6721 aaattgtatt tgttaaatat gtactacaaa cttagtagtt ggaagggcta attcactccc 6781 aaagaagaca agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt 6841 agcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga tggtgctaca 6901 agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag aacaccagct 6961 tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagtg ttagagtgga 7021 ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg gagtacttca 7081 agaactgctg atatcgagct tgctacaagg gactttccgc tggggacttt ccagggaggc 7141 gtggcctggg cgggactggg gagtggcgag ccctcagatc ctgcatataa gcagctgctt 7201 tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa 7261 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt 7321 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg 7381 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc 7441 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac 7501 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga 7561 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag 7621 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat 7681 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc 7741 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa 7801 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga 7861 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccggc cgctgatctt 7921 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 7981 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 8041 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 8101 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 8161 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 8221 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 8281 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 8341 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg 8401 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga 8461 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag 8521 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat 8581 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt 8641 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga 8701 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt 8761 agtgaacgga tctcgacggt atcgccttta aaagaaaagg ggggattggg gggtacagtg 8821 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac 8881 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttatc 8941 gatgagtaat tcatacaaaa ggactcgccc ctgccttggg gaatcccagg gaccgtcgtt 9001 aaactcccac taacgtagaa cccagagatc gctgcgttcc cgccccctca cccgcccgct 9061 ctcgtcatca ctgaggtgga gaagagcatg cgtgag //