LOCUS Exported 6223 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION protein expression in E. coli. ACCESSION . VERSION . KEYWORDS pET32a-Spike-S-4P SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6223) AUTHORS Shen H, Forgacs D, Chapla D, Moremen KW, Wells L, Hamer SA, Tompkins SM, Ross TM, Rouphael N, Edupuganti S, Collins MH, Tarleton RL. TITLE A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses. JOURNAL medRxiv 2021.(pre-print) 01.20.21249279 REFERENCE 2 (bases 1 to 6223) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6223 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 12..467 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(99..118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 309..330 /label=F1ori-F /note="F1 origin, forward primer" promoter 494..598 /gene="bla" /label=AmpR promoter CDS 599..1459 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(817..836) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1630..2218 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2119..2138 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2372..2389 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2404..2546 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2472..2491) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2632..2654 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2648..2839) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3648..4730) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 4692..4711 /label=LacI-R /note="LacI, reverse primer" promoter complement(4731..4808) /gene="lacI" /label=lacI promoter primer_bind 5014..5033 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5121..5140 /label=T7 /note="T7 promoter, forward primer" promoter 5121..5139 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5140..5164 /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)." RBS 5179..5201 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5209..5535 /codon_start=1 /gene="trxA" /product="E. coli thioredoxin" /label=TrxA /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL DANLA" CDS 5557..5574 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5584..5601 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 5608..5652 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 5668..5682 /codon_start=1 /product="enterokinase recognition and cleavage site" /label=enterokinase site /translation="DDDDK" CDS 6067..6084 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6137..6155) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6151..6198 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1141 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1741 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2401 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2461 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2521 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2581 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2641 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2701 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2761 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2821 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2881 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2941 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3001 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3061 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3121 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3181 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3241 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3301 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3361 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3421 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3481 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3541 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3601 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3661 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3721 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3781 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3841 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3901 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3961 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4021 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4081 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4141 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4201 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4261 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4321 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4381 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4441 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4501 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4561 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4621 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4681 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4741 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4801 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4861 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4921 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4981 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5041 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5101 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5161 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gagcgataaa 5221 attattcacc tgactgacga cagttttgac acggatgtac tcaaagcgga cggggcgatc 5281 ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat 5341 gaaatcgctg acgaatatca gggcaaactg accgttgcaa aactgaacat cgatcaaaac 5401 cctggcactg cgccgaaata tggcatccgt ggtatcccga ctctgctgct gttcaaaaac 5461 ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag gtcagttgaa agagttcctc 5521 gacgctaacc tggccggttc tggttctggc catatgcacc atcatcatca tcattcttct 5581 ggtctggtgc cacgcggttc tggtatgaaa gaaaccgctg ctgctaaatt cgaacgccag 5641 cacatggaca gcccagatct gggtaccgac gacgacgaca aggccatggc tgatatcgga 5701 tccatgaaaa agacggggac aggagtacta actgagagca ataaaaaatt cctgccgttt 5761 cagcaatttg gtcgcgatat tgcaggctct atgaagaagt tcgctcaggt taaacaaatc 5821 tataaaaccc cgcctattaa agacttcggc ggttttaact tcagccagat cggctctatg 5881 aagaagccag atccgtcgaa gccgtccaaa cgtagcttta tcgaagatct gctgtttaac 5941 aaggtgacgc tggcggatgg ttccatgaag aagtacgacc cgctccaacc ggaattggac 6001 agcttcaaag aggagttgga caaatacttc aagaaccaca ccagcaagct tgcggccgca 6061 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct 6121 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 6181 gtcttgaggg gttttttgct gaaaggagga actatatccg gat //