LOCUS Exported 7222 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 endoRNAse. ACCESSION . VERSION . KEYWORDS pGBW-m4134125 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7222) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7222) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7222 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1040..1060 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1061..1138 /codon_start=1 /product="calmodulin-binding peptide" /label=CBP /note="derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium" /translation="KRRWKKNFIAVSAANRFKKISSSGAL" polyA_signal 1191..1312 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1202..1221) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1256..1275 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1427..1473 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1503..1639 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1684..1755 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1981..2569) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2061..2080) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3069..4278 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4115..4134 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4282..4881 /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(4282..4301) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4778..4798 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4908..4956 /note="synthetic polyadenylation signal" CDS 5222..6082 /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 complement(5440..5459) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6095..6231 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6376..6447 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6452..6498 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6516..6564 /note="synthetic polyadenylation signal" enhancer 6645..6948 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6950..7152 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7103..7123 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgtta gaaaatgtgg cttttaatgt tgtaaataag ggacactttg atggacaaca 61 gggtgaagta ccagtttcta tcattaataa cactgtttac acaaaagttg atggtgttga 121 tgtagaattg tttgaaaata aaacaacatt acctgttaat gtagcatttg agctttgggc 181 taagcgcaac attaaaccag taccagaggt gaaaatactc aataatttgg gtgtggacat 241 tgctgctaat actgtgatct gggactacaa aagagatgct ccagcacata tatctactat 301 tggtgtttgt tctatgactg acatagccaa gaaaccaact gaaacgattt gtgcaccact 361 cactgtcttt tttgatggta gagttgatgg tcaagtagac ttatttagaa atgcccgtaa 421 tggtgttctt attacagaag gtagtgttaa aggtttacaa ccatctgtag gtcccaaaca 481 agctagtctt aatggagtca cattaattgg agaagccgta aaaacacagt tcaattatta 541 taagaaagtt gatggtgttg tccaacaatt acctgaaact tactttactc agagtagaaa 601 tttacaagaa tttaaaccca ggagtcaaat ggaaattgat ttcttagaat tagctatgga 661 tgaattcatt gaacggtata aattagaagg ctatgccttc gaacatatcg tttatggaga 721 ttttagtcat agtcagttag gtggtttaca tctactgatt ggactagcta aacgttttaa 781 ggaatcacct tttgaattag aagattttat tcctatggac agtacagtta aaaactattt 841 cataacagat gcgcaaacag gttcatctaa gtgtgtgtgt tctgttattg atttattact 901 tgatgatttt gttgaaataa taaaatccca agatttatct gtagtttcta aggttgtcaa 961 agtgactatt gactatacag aaatttcatt tatgctttgg tgtaaagatg gccatgtaga 1021 aacattttac ccaaaattag aaaaccttta ctttcagtcg aagaggcgat ggaaaaagaa 1081 ttttatcgcg gtgagtgcag ccaacagatt caaaaagata tcctcgtctg gtgccctcta 1141 agctagcttg actgactgag atacagcgta ccttcagctc acagacatga taagatacat 1201 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 1261 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 1321 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa 1381 gtaaaacctc tacaaatgtg gtattggccc atctctatcg gtatcgtagc ataacccctt 1441 ggggcctcta aacgggtctt gaggggtttt ttgtgcccct cgggccggat tgctatctac 1501 cggcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat 1561 gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt 1621 accagaaatt tatccttaag gtcgtcagct atcctgcagg cgatctctcg atttcgatca 1681 agacattcct ttaatggtct tttctggaca ccactagggg tcagaagtag ttcatcaaac 1741 tttcttccct ccctaatctc attggttacc ttgggctatc gaaacttaat taaccagtca 1801 agtcagctac ttggcgagat cgacttgtct gggtttcgac tacgctcaga attgcgtcag 1861 tcaagttcga tctggtcctt gctattgcac ccgttctccg attacgagtt tcatttaaat 1921 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 1981 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 2041 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 2101 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 2161 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 2221 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 2281 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 2341 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 2401 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 2461 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 2521 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 2581 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 2641 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 2701 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 2761 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta aatttccgaa 2821 ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc ttgcaggcta 2881 cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct attgcttggc 2941 agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat cacccgagag 3001 aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat cgaaatcggg 3061 gcgcgcctgg cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc tcgtcacggc 3121 gagcgctgcc acgtcagacg aagggcgcag gagcgtcctg atccttccgc ccggacgctc 3181 aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca gcagaaggac 3241 attttaggac gggacttggg tgactctagg gcactggttt tctttccaga gagcggaaca 3301 ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg gggcggtgaa 3361 cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc gcagccggga 3421 tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag tagcgggctg 3481 ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag 3541 agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact gggggttggg 3601 gggagcgcag caaaatggcg gctgttcccg agtcttgaat ggaagacgct tgtgaggcgg 3661 gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc caaggtcttg 3721 agcccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggc accatctggg 3781 gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg ttgcgggggc 3841 ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc gcgccctcgt 3901 cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca cctgccggta 3961 ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc 4021 ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg tcagtttctt 4081 tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg aactatgcgc 4141 tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt 4201 gggtcaatat gtaattttca gtgttagact tgtaaattgt ccgctaaatt ctggccgttt 4261 ttggcttttt tgttagacaa catgaccgag tacaagccca cggtgcgcct cgccacccgc 4321 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg 4381 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc 4441 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg 4501 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg 4561 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga gggcctcctg 4621 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac 4681 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc cgccgagcgc 4741 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg 4801 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg 4861 acccgcaagc ccggtgccta ggctagtatg taagcctagt cttagataat aaaatcgcta 4921 tccatcgaag atggatgtgt gttggttttt tgtgtgtgta acgctaggcg cgcctggtgt 4981 accgagaacg atcctctcag tgcgagtctc gacgatccat atcgttgctt ggcagtcagc 5041 cagtcggaat ccagcttggg acccaggaag tccaatcgtc agatattgta ctcaagcctg 5101 gtcacggcag cgtaccgatc tgtttaaacc tagatattga tagtctgatc ggtcaacgta 5161 taatcgagtc ctagcttttg caaacatcta tcaagagaca ggatcagcag gaggctttcg 5221 catgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 5281 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 5341 gcgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 5401 cgaagaacgc tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 5461 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 5521 ggttgagtat tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 5581 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 5641 tggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 5701 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 5761 gcctgtagca atggcaacaa ccttgcgtaa actattaact ggcgaactac ttactctagc 5821 ttcccggcaa cagttgatag actggatgga ggcggataaa gttgcaggac cacttctgcg 5881 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 5941 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 6001 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 6061 ctcactgatt aagcattggt aaccgattct aggtgcattg gcgcagaaaa aaatgcctga 6121 tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc 6181 ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta agatcgttta 6241 aactcgactc tggctctatc gaatctccgt cgtttcgagc ttacgcgaac agccgtggcg 6301 ctcatttgct cgtcgggcat cgaatctcgt cagctatcgt cagcttacct ttttggcagc 6361 gatcgcggct cccgacatct tggaccatta gctccacagg tatcttcttc cctctagtgg 6421 tcataacagc agcttcagct acctctcaat tcaaaaaacc cctcaagacc cgtttagagg 6481 ccccaagggg ttatgctatc aatcgttgcg ttacacacac aaaaaaccaa cacacatcca 6541 tcttcgatgg atagcgattt tattatctaa ctgctgatcg agtgtagcca gatctagtaa 6601 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 6661 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 6721 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 6781 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 6841 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 6901 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt 6961 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 7021 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 7081 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 7141 ataagcagag ctggtttagt gaaccgtcag atcagatctt tgtcgatcct accatccact 7201 cgacacaccc gccagcggcc gc //