LOCUS       sequence-324312-        5570 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 5570)
  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 5570)
  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: pET30a-SARS-CoV2-ORF3b (S23Q)
FEATURES             Location/Qualifiers
     source          1..5570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     958..977
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(997..1074)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1280..1299
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1387..1406
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1387..1405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1406..1430
                     /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             1445..1467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1478..1495
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1505..1522
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1529..1573
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1589..1603
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             1817..1834
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1887..1905)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1901..1948
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1985..2440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2072..2091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2282..2303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2532..3347)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2532..3347)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3255..3274
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3339..3358
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3469..4057
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3958..3977
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4211..4228
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4243..4382
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4308..4327)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4468..4490
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4484..4675)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5484..5570,1..996))
                     /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"
ORIGIN
        1 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
       61 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
      121 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
      181 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
      241 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
      301 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
      361 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
      421 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
      481 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
      541 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
      601 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
      661 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
      721 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
      781 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
      841 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
      901 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
      961 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     1021 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     1081 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     1141 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     1201 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     1261 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     1321 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     1381 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     1441 taattttgtt taactttaag aaggagatat acatatgcac catcatcatc atcattcttc
     1501 tggtctggtg ccacgcggtt ctggtatgaa agaaaccgct gctgctaaat tcgaacgcca
     1561 gcacatggac agcccagatc tgggtaccga cgacgacgac aaggccatgg ctgatatcgg
     1621 atccatgatg ccgaccatct tctttgcggg tatcctgatt gtgaccacca tcgtttacct
     1681 gaccattgtg cagctgctgc aactgagcct gctgcaggtg atggcgcagc aagttctgtt
     1741 cctgaacatg accacccgtc tggtggttat tctgaagaac ggcaacctgg agtaaaagct
     1801 tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     1861 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1921 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1981 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2041 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2101 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2161 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2221 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2281 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2341 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2401 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     2461 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2521 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2581 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2641 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2701 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2761 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2821 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2881 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2941 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     3001 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     3061 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     3121 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     3181 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     3241 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     3301 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     3361 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     3421 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3481 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3541 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3601 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3661 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3721 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3781 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3841 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3901 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3961 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4021 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4081 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4141 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4201 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4261 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     4321 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     4381 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4441 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4501 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4561 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4621 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4681 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4741 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4801 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4861 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4921 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4981 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     5041 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     5101 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     5161 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     5221 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     5281 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     5341 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     5401 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     5461 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5521 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
//
