LOCUS Exported 7714 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PiggybacTransposon-based tunable and temporal expression control of SARS-CoV2 N and mCherry. ACCESSION . VERSION . KEYWORDS XLone-BSD SARS-CoV2 N P2A mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7714) TITLE Inducible COVID19 SARS-CoV2 Nucleocapsid Expressing Plasmid in Mammalian Cells JOURNAL Unpublished REFERENCE 2 (bases 1 to 7714) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7714 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 9..376 /label=TRE3GS promoter /note="3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On(R) 3G, modified to eliminate binding sites for endogenous mammalian transcription factors" protein_bind 17..35 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 53..71 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 89..107 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 125..143 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 161..179 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 197..215 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 233..251 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 1679..1735 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 1739..1762 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 1763..2470 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(1905..1923) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2173..2192 /label=mCherry-F /note="mCherry, forward primer" polyA_signal 2676..2810 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" repeat_region 3086..3120 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(3131..3148) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3131..3147) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3140..3162) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(3356..3375) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3475..3497 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3535..3553) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3621..3725 /gene="bla" /label=AmpR promoter CDS 3726..4586 /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(3944..3963) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4757..5345 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5246..5265 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5499..5516 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5633..5654 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5669..5699 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5707..5723 /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)." primer_bind 5712..5734 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5731..5747 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5731..5747 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" repeat_region 5773..5835 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" polyA_signal 6011..6092 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" CDS complement(6209..6607) /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" CDS complement(6608..6664) /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS complement(6674..7417) /codon_start=1 /product="modified rtTA protein that binds tightly to promoters containing the tet operator in the presence of doxycycline" /label=Tet-On(R) 3G /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" CDS complement(7418..7441) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 7441..7450 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(7470..7681) /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" ORIGIN 1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc 61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt 121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa 181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc 241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg 301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac 361 ttcctaccct cgtaaaggta ccgccaccat gagcgataac ggcccccaaa accaacggaa 421 tgcccctcgg ataacatttg gtggccctag tgattccaca ggaagtaacc aaaacggaga 481 acggtctgga gcccgctcta aacagaggcg accacaggga ttgccaaata acacagcgtc 541 atggtttaca gccctgactc aacatggaaa ggaggacttg aaatttccac gaggacaggg 601 tgtaccaata aacacaaaca gtagtccaga cgatcagatc gggtattata gaagggccac 661 gagacgcatc agaggtggag atggtaaaat gaaagatctc agtccgcgct ggtactttta 721 ttatttggga accggcccag aagcgggcct cccatatgga gccaataagg acgggattat 781 atgggtggct acagagggag ccctcaatac gccaaaagat cacatcggaa caagaaaccc 841 tgctaataat gccgcaatcg tgctgcagtt gcctcagggg acgacgcttc ctaaaggctt 901 ttacgcagaa ggatcacgcg gcggcagcca agcatcctca aggtctagtt ccagaagtcg 961 aaacagctcc agaaactcca caccagggtc cagtaggggc acaagtccgg cgcggatggc 1021 gggcaacggc ggagacgccg cactcgctct gttgctcctg gaccggctca accaacttga 1081 atccaagatg agtggtaagg gacagcagca acaaggtcaa accgtaacca agaaaagcgc 1141 tgcagaagct tctaaaaaac ctcgacaaaa acggaccgct acgaaggcat ataacgttac 1201 tcaagccttc gggagacgag ggccggagca aacccagggg aattttggag atcaggagct 1261 catccggcaa gggacagatt acaaacattg gcctcaaatc gctcagttcg cgccgtccgc 1321 aagtgctttt ttcggaatgt ctcgcatcgg tatggaagta acaccatccg ggacatggct 1381 cacttatacc ggcgccatca agctggacga caaagatcca aatttcaaag atcaggtgat 1441 tctcttgaat aaacacatag acgcgtataa gacgtttcca cccaccgaac ccaagaagga 1501 caaaaagaaa aaagctgatg aaacacaagc gttgccccag cgacagaaga agcagcaaac 1561 cgtcaccctc ctgccagccg ctgatctcga tgacttctcc aagcaacttc aacagtccat 1621 gagctcagcc gattctactc aggcgctcga aggcggcggg ggagctagcg gcagcggcgc 1681 cactaacttc tccctgttga aacaagcagg ggatgtcgaa gagaatcccg ggccaatgga 1741 ctacaaggac gacgatgaca aggtgagcaa gggcgaggag gataacatgg ccatcatcaa 1801 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat 1861 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac 1921 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc 1981 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga 2041 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca 2101 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt 2161 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg 2221 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga 2281 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct 2341 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac 2401 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct 2461 gtacaagtaa gaattccgat catattcaat aacccactag tagaccacct cccctgcgag 2521 ctaagctgga cagccaatga cgggtaagag agtgacattt ttcactaacc taagacagga 2581 gggccgtcag agctactgcc taatccaaag acgggtaaaa gtgataaaaa tgtatcactc 2641 caacctaaga caggcgcagc ttccgaggga tttgagatcc agacatgata agatacattg 2701 atgagtttgg acaaaccaaa actagaatgc agtgaaaaaa atgccttatt tgtgaaattt 2761 gtgatgctat tgccttattt gtaaccatta taagctgcaa taaacaagtt tgatatctat 2821 aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa 2881 ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc 2941 acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg 3001 gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga 3061 aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg 3121 ttaagaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 3181 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 3241 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt 3301 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 3361 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 3421 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 3481 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 3541 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 3601 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 3661 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 3721 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 3781 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 3841 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 3901 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 3961 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 4021 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 4081 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 4141 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 4201 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 4261 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 4321 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 4381 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 4441 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 4501 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 4561 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 4621 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 4681 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 4741 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 4801 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 4861 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 4921 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 4981 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 5041 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 5101 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 5161 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 5221 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 5281 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 5341 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 5401 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 5461 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 5521 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 5581 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 5641 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 5701 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 5761 aggtcgactt aaccctagaa agataatcat attgtgacgt acgttaaaga taatcatgcg 5821 taaaattgac gcatgtgttt tatcggtctg tatatcgagg tttatttatt aatttgaata 5881 gatattaagt tttattatat ttacacttac atactaataa taaattcaac aaacaattta 5941 tttatgttta tttatttatt aaaaaaaaac aaaaactcaa aatttcttct ataaagtaac 6001 aaaactttta gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat 6061 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt 6121 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg 6181 gtatggctga ttatgatcct ctggagattt agccctccca cacataacca gagggcagca 6241 attcacgaat cccaactgcc gtcggctgtc catcactgtc cttcactatg gctttgatcc 6301 caggatgcag atcgagaagc acctgtcggc accgtccgca ggggctcaag atgcccctgt 6361 tctcatttcc gatcgcgacg atacaagtca ggttgccagc tgccgcagca gcagcagtgc 6421 ccagcaccac gagttctgca caaggtcccc cagtaaaatg atatacattg acaccagtga 6481 agatgcggcc gtcgctagag agagctgcgc tggcgacgct gtagtcttca gagatgggga 6541 tgctgttgat tgtagccgtt gctctttcaa tgagggtgga ttcttcttga gacaaaggct 6601 tggccatggg gccggggttc tcctccacgt cgccggcctg cttcagcagg ctgaagttgg 6661 tggcgccgct gcccccgggg agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca 6721 gcatatcaag gtcaaagtcg tcaagggcat cggctgggag catgtctaag tcaaaatcgt 6781 caagggcgtc ggtcggcccg ccgctttcgc actttagctg tttctccagg ccacatatga 6841 ttagttccag gccgaaaagg aaggcaggtt cggctccctg ccggtcgaac agctcaattg 6901 cttgtttcag aagtgggggc atagaatcgg tggtaggtgt ctctctttcc tcttttgcta 6961 cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa gtggcccacg gcggacagag 7021 cgtacagtgc gttctccagg gagaagcctt gctgacacag gaacgcgagc tgattttcca 7081 gggtttcgta ctgtttctct gttgggcggg tgccgagatg cactttagcc ccgtcgcgat 7141 gtgagaggag agcacagcgg tatgacttgg cgttgttccg cagaaagtct tgccatgact 7201 cgccttccag ggggcaggag tgggtatgat gcctgtccag catctcgatt ggcagggcat 7261 cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt aggctgctca actcccagct 7321 tttgagcgag tttccttgtc gtcaggcctt cgataccgac tccattgagt aattccagag 7381 cagagtttat gactttgctc ttgtccagtc tagacatctt atcgtcatcg tctttgtaat 7441 ccatggtggc ggatcccgcg tcacgacacc tgtgttctgg cggcaaaccc gttgcgaaaa 7501 agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg 7561 cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg 7621 gttcaattgc cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc 7681 cactgacggg caccggagcc actcgagtgg aatt //