LOCUS Exported 9333 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-nsp10-2xStrep-IRES-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9333) 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 9333) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9333 /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" regulatory 3544..3553 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3988..4011 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 4048..4071 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" misc_feature 4083..4656 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4250..4267) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4477..4496 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 4689..5288 /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(4689..4708) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5185..5205 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 5302..5890 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5355..5375) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5773..5784) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6097..6730 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" primer_bind complement(6858..6874) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6858..6874) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6871..6893) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6882..6898 /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(6906..6936) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6951..6972 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7089..7106) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7260..7848) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7340..7359) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8019..8879) /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 8642..8661 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8880..8984) /gene="bla" /label=AmpR promoter polyA_signal 9032..9166 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(9069..9088) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 9123..9142 /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 tggctggtaa tgcaactgag gttcccgcga atagtacggt tctgagcttt 3601 tgcgcctttg ctgttgacgc agctaaggca tataaggact acttggcctc cgggggccag 3661 ccaatcacaa actgcgtcaa gatgttgtgt acgcacacgg gcacaggtca ggcaatcact 3721 gtcacacctg aagctaacat ggatcaggaa tcttttggtg gtgccagttg ttgtctgtac 3781 tgtcgatgtc atatagatca tcccaacccg aaagggtttt gtgatctgaa aggaaaatac 3841 gtccaaatcc cgaccacgtg cgcgaacgac ccggttgggt ttacgttgaa aaataccgta 3901 tgtactgtct gtggtatgtg gaagggctat ggctgttcat gcgatcaatt gcgagagccg 3961 atgttgcagc tcgaaggcgg cgggggatgg agccatccac aattcgagaa aggcggtggt 4021 tcaggaggag gtagcggggg tggatcatgg tcacatccgc agtttgaaaa gtaaggatcc 4081 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 4141 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 4201 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 4261 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 4321 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 4381 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 4441 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 4501 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 4561 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 4621 ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaaccc acaaggagac 4681 gaccttccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc 4741 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg 4801 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg 4861 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca 4921 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt 4981 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc 5041 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg 5101 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg 5161 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg 5221 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg 5281 gtgcctagac gcgtctggaa caatcaacct ctggattaca aaatttgtga aagattgact 5341 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 5401 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 5461 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 5521 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 5581 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 5641 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg 5701 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc 5761 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 5821 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc 5881 gcctccccgc ctggaattaa ttctgcagtc gagacctaga aaaacatgga gcaatcacaa 5941 gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg 6001 aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg 6061 tagatcttag ccacttttta aaagaaaaga ggggactgga agggctaatt cactcccaac 6121 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattagc 6181 agaactacac accagggcca ggggtcagat atccactgac ctttggatgg tgctacaagc 6241 tagtaccagt tgagccagat aaggtagaag aggccaataa aggagagaac accagcttgt 6301 tacaccctgt gagcctgcat gggatggatg acccggagag agaagtgtta gagtggaggt 6361 ttgacagccg cctagcattt catcacgtgg cccgagagct gcatccggag tacttcaaga 6421 actgctgata tcgagcttgc tacaagggac tttccgctgg ggactttcca gggaggcgtg 6481 gcctgggcgg gactggggag tggcgagccc tcagatcctg catataagca gctgcttttt 6541 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta 6601 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc 6661 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa 6721 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa 6781 tgaatatcag agagtgagag gccttgacat tgctagcgtt taccgtcgac ctctagctag 6841 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 6901 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 6961 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 7021 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 7081 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 7141 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 7201 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 7261 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 7321 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 7381 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 7441 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 7501 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 7561 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 7621 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 7681 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 7741 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 7801 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 7861 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 7921 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 7981 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 8041 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 8101 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 8161 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 8221 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 8281 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 8341 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 8401 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 8461 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 8521 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 8581 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 8641 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 8701 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 8761 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 8821 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 8881 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 8941 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 9001 gtgccacctg acgtcgacgg atcgggagat caacttgttt attgcagctt ataatggtta 9061 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 9121 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca 9181 agctaaccaa aatcatccca aacttcccac cccataccct attaccactg ccaattacct 9241 gtggtttcat ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg 9301 tatttgttaa atatgtacta caaacttagt agt //