LOCUS       sequence-324363-        7076 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 7076)
  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 7076)
  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-FluB-NP
FEATURES             Location/Qualifiers
     source          1..7076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1291..1310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1398..1417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1398..1416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /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             1456..1478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1489..1506
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1516..1533
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1540..1584
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1600..1614
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             3100..3111
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3334..3351
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3404..3422)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3418..3465
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3502..3957
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3589..3608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3799..3820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4049..4864)
                     /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(4049..4864)
                     /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     4772..4791
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4856..4875
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4986..5574
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5475..5494
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5728..5745
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5760..5899
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5825..5844)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5985..6007
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6001..6192)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7001..7076,1..1007))
                     /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 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga
     1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1441 ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgca ccatcatcat
     1501 catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa
     1561 ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg
     1621 gctgatatcg gatccatgag caacatggac attgatggta tgaacaccgg caccattgac
     1681 aagaccccgg aggaaatcac cagcggtacc agcggtacca cccgtccgat cattcgtccg
     1741 gcgaccctgg cgccgccgag caacaagcgt acccgtaacc cgagcccgga gcgtgcgacc
     1801 accagcagcg aagacgatgt tggtcgtaaa gcgcagaaga aacaaacccc gaccgagatc
     1861 aagaaaagcg tgtacaacat ggtggttaag ctgggtgaat tctataacca gatgatggtt
     1921 aaagcgggcc tgaacgacga tatggagcgt aacctgattc aaaacgcgca cgcggtggaa
     1981 cgtatcctgc tggcggcgac cgacgataag aaaaccgagt ttcagaagaa aaagaacgcg
     2041 cgtgacgtga aagaaggcaa ggaggaaatt gatcacaaca agaccggtgg caccttctac
     2101 aagatggttc gtgacgataa aaccatctat tttagcccga tccgtattac cttcctgaaa
     2161 gaggaagtga agaccatgta caaaaccacc atgggcagcg acggttttag cggcctgaac
     2221 cacatcatga ttggccacag ccagatgaac gacgtgtgct tccaacgtag caaagcgctg
     2281 aagcgtgttg gtctggaccc gagcctgatt agcacctttg cgggcagcac cgttccgcgt
     2341 cgtagcggtg cgaccggtgt ggcgatcaaa ggtggcggta ccctggttgc ggaagcgatt
     2401 cgtttcatcg gtcgtgcgat ggcggaccgt ggcctgctgc gtgatattaa agcgaagacc
     2461 gcgtatgaga agatcctgct gaacctgaaa aacaagtgca gcgcgccgca gcaaaaagcg
     2521 ctggttgatc aggtgatcgg tagccgtaac ccgggcattg cggacatcga agatctgacc
     2581 ctgctggcgc gtagcatggt ggttgtgcgt ccgagcgttg cgagcaaggt tgtgctgccg
     2641 atcagcattt acgcgaaaat tccgcagctg ggttttaacg ttgaggaata cagcatggtg
     2701 ggctatgagg cgatggcgct gtataacatg gcgaccccgg ttagcatcct gcgtatgggt
     2761 gacgatgcga aagacaagag ccaactgttc tttatgagct gcttcggcgc ggcgtacgag
     2821 gatctgcgtg tgctgagcgc gctgaccggt accgaattta aaccgcgtag cgcgctgaaa
     2881 tgcaagggct tccacgttcc ggcgaaggag caggtggaag gcatgggtgc ggcgctgatg
     2941 agcattaaac tgcagttttg ggcgccgatg acccgtagcg gtggtaacga ggcgggtggt
     3001 gacggtggta gcggtcagat tagctgcagc ccggttttcg cggtggaacg tccgatcgcg
     3061 ctgagcaagc aagcggtgcg tcgtatgctg agcatgaaca tcgagggtcg tgacgcggat
     3121 gttaagggca acctgctgaa aatgatgaac gacagcatgg cgaaaaagac cagcggtaac
     3181 gcgtttattg gcaaaaagat gttccagatc agcgataaaa acaagaccaa cccgatcgag
     3241 attccgatca aacaaaccat cccgaacttc tttttcggtc gtgacaccgc ggaagattat
     3301 gacgatctgg ataagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     3361 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     3421 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     3481 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3541 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3601 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3661 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3721 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3781 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3841 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3901 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     3961 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4021 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     4081 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     4141 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     4201 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     4261 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     4321 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     4381 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     4441 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     4501 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     4561 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     4621 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     4681 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     4741 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     4801 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     4861 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     4921 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4981 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5041 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5101 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     5161 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5221 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5281 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5341 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5401 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5461 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5521 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5581 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5641 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5701 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5761 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     5821 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     5881 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5941 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     6001 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     6061 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     6121 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     6181 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     6241 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     6301 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     6361 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     6421 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     6481 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     6541 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     6601 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     6661 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     6721 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     6781 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     6841 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     6901 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     6961 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     7021 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcg
//
