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" primer_bind 38..57 /label=T7 /note="T7 promoter, forward primer" promoter 38..56 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 57..81 /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 96..118 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 126..452 /codon_start=1 /gene="trxA" /product="E. coli thioredoxin" /label=TrxA /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL DANLA" CDS 474..491 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 501..518 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 525..569 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 585..599 /codon_start=1 /product="enterokinase recognition and cleavage site" /label=enterokinase site /translation="DDDDK" CDS 984..1001 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1054..1072) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1068..1115 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1152..1607 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1239..1258) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1449..1470 /label=F1ori-F /note="F1 origin, forward primer" promoter 1634..1738 /gene="bla" /label=AmpR promoter CDS 1739..2599 /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(1957..1976) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2770..3358 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3259..3278 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3512..3529 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3544..3686 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3612..3631) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3772..3794 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3788..3979) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4788..5870) /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 5832..5851 /label=LacI-R /note="LacI, reverse primer" promoter complement(5871..5948) /gene="lacI" /label=lacI promoter primer_bind 6154..6173 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa 61 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata 121 tacatatgag cgataaaatt attcacctga ctgacgacag ttttgacacg gatgtactca 181 aagcggacgg ggcgatcctc gtcgatttct gggcagagtg gtgcggtccg tgcaaaatga 241 tcgccccgat tctggatgaa atcgctgacg aatatcaggg caaactgacc gttgcaaaac 301 tgaacatcga tcaaaaccct ggcactgcgc cgaaatatgg catccgtggt atcccgactc 361 tgctgctgtt caaaaacggt gaagtggcgg caaccaaagt gggtgcactg tctaaaggtc 421 agttgaaaga gttcctcgac gctaacctgg ccggttctgg ttctggccat atgcaccatc 481 atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa accgctgctg 541 ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac gacgacaagg 601 ccatggctga tatcggatcc atgaaaaaga cggggacagg agtactaact gagagcaata 661 aaaaattcct gccgtttcag caatttggtc gcgatattgc aggctctatg aagaagttcg 721 ctcaggttaa acaaatctat aaaaccccgc ctattaaaga cttcggcggt tttaacttca 781 gccagatcgg ctctatgaag aagccagatc cgtcgaagcc gtccaaacgt agctttatcg 841 aagatctgct gtttaacaag gtgacgctgg cggatggttc catgaagaag tacgacccgc 901 tccaaccgga attggacagc ttcaaagagg agttggacaa atacttcaag aaccacacca 961 gcaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca 1021 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc 1081 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat 1141 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 1201 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 1261 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 1321 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 1381 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 1441 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 1501 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 1561 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 1621 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 1681 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 1741 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 1801 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 1861 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 1921 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 1981 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 2041 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 2101 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 2161 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 2221 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 2281 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 2341 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 2401 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 2461 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 2521 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 2581 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 2641 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 2701 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 2761 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 2821 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 2881 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 2941 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 3001 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 3061 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 3121 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 3181 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 3241 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 3301 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 3361 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 3421 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 3481 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 3541 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 3601 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 3661 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 3721 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 3781 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 3841 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 3901 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 3961 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 4021 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 4081 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 4141 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 4201 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 4261 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 4321 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 4381 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 4441 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 4501 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 4561 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 4621 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 4681 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 4741 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 4801 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 4861 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 4921 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 4981 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 5041 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 5101 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 5161 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 5221 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 5281 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 5341 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 5401 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 5461 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 5521 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 5581 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 5641 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 5701 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 5761 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 5821 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 5881 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 5941 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 6001 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 6061 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 6121 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 6181 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccg //