LOCUS Exported 5693 bp ds-DNA circular SYN 14-OCT-2021 DEFINITION Expresses K164A K165A CoV2 nsp1 in mammalian cells. This contains a C-terminal 3xFLAG tag fusion to nsp1.. ACCESSION . VERSION . KEYWORDS pCDNA4TO K164A H165A CoV-2 nsp1 3xFLAG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5693) AUTHORS Glaunsinger, BA. TITLE The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression. JOURNAL Unpublished REFERENCE 2 (bases 1 to 5693) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 14, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5693 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 820..838 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 841..859 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 869..893 /label=LNCX /note="Human CMV promoter, forward primer" CDS 1623..1688 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" primer_bind complement(1714..1731) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1720..1944 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 1990..2418 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2077..2096) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2287..2308 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2427..2447) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2432..2761 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2612..2747 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2674..2693 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2862..3236 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 3366..3487 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3403..3422) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3457..3476 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3536..3552) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3536..3552) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3549..3571) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3560..3576 /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(3584..3614) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3629..3650 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3767..3784) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3938..4526) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4018..4037) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4697..5557) /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 5320..5339 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5558..5662) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc 841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga 901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga 961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga 1021 attctgcaga tatccagcac agtggcggcc gcatggagag ccttgtccct ggtttcaacg 1081 agaaaacaca cgtccaactc agtttgcctg ttttacaggt tcgcgacgtg ctcgtacgtg 1141 gctttggaga ctccgtggag gaggtcttat cagaggcacg tcaacatctt aaagatggca 1201 cttgtggctt agtagaagtt gaaaaaggcg ttttgcctca acttgaacag ccctatgtgt 1261 tcatcaaacg ttcggatgct cgaactgcac ctcatggtca tgttatggtt gagctggtag 1321 cagaactcga aggcattcag tacggtcgta gtggtgagac acttggtgtc cttgtccctc 1381 atgtgggcga aataccagtg gcttaccgca aggttcttct tcgtaagaac ggtaataaag 1441 gagctggtgg ccatagttac ggcgccgatc taaagtcatt tgacttaggc gacgagcttg 1501 gcactgatcc ttatgaagat tttcaagaaa actggaacac tgcagctagc agtggtgtta 1561 cccgtgaact catgcgtgag cttaacggag ggtggcggcc gctcgaggga ggcggtggag 1621 ccgactacaa ggaccacgac ggcgactaca aggaccacga catcgactac aaggacgacg 1681 acgacaaggg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 1741 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 1801 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 1861 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 1921 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg 1981 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2041 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2101 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2161 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 2221 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 2281 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 2341 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 2401 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 2461 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 2521 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 2581 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 2641 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 2701 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 2761 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa 2821 tcatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg 2881 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc 2941 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga 3001 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt 3061 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc 3121 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg 3181 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg 3241 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 3301 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 3361 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 3421 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 3481 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat 3541 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 3601 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 3661 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 3721 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 3781 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3841 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3901 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 3961 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4021 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4081 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 4141 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 4201 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 4261 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 4321 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 4381 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 4441 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 4501 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 4561 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 4621 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 4681 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 4741 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 4801 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 4861 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 4921 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 4981 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 5041 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 5101 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 5161 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 5221 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 5281 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 5341 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 5401 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 5461 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 5521 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 5581 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 5641 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc //