LOCUS       sequence-325882-        6223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 [Preprint]. 2021 Jan 27:2021.01.20.21249279. doi: 
            10.1101/2021.01.20.21249279.
  PUBMED    33532799
REFERENCE   2  (bases 1 to 6223)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "medRxiv 
            [Preprint]. 2021 Jan 27:2021.01.20.21249279. doi: 
            10.1101/2021.01.20.21249279."
COMMENT     Sequence Label: pET32a-Spike-S-4P
FEATURES             Location/Qualifiers
     source          1..6223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             join(1739..1807,1808..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
//
