LOCUS       sequence-324362-        6893 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 6893)
  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 6893)
  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-FluA-NP
FEATURES             Location/Qualifiers
     source          1..6893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             978..995
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1048..1066)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1062..1109
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1146..1601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1233..1252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1443..1464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1693..2508)
                     /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(1693..2508)
                     /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     2416..2435
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2500..2519
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2630..3218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3119..3138
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3372..3389
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3404..3543
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3469..3488)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3629..3651
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3645..3836)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4645..5727)
                     /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     5689..5708
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5728..5805)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6011..6030
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6118..6137
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6118..6136
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6137..6161
                     /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             6176..6198
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6209..6226
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6236..6253
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6260..6304
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             6320..6334
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
ORIGIN
        1 gtgcggcggt gaagggtgtt ggcaccatcg cgatggagct gatccgtatg attaaacgtg
       61 gtattaacga tcgtaacttc tggcgtggcg agaacggccg tcgtacccgt gtggcgtatg
      121 aacgtatgtg caacatcctg aagggcaaat ttcagaccgc ggcgcaacgt gcgatgatgg
      181 accaggttcg tgaaagccgt aacccgggta acgcggagat cgaagatctg attttcctgg
      241 cgcgtagcgc gctgattctg cgtggcagcg tggcgcacaa gagctgcctg ccggcgtgcg
      301 tgtacggtct ggcggttgcg agcggtcacg acttcgagcg tgaaggttat agcctggttg
      361 gcatcgatcc gtttaagctg ctgcagaaca gccaagtggt tagcctgatg cgtccgaacg
      421 agaacccggc gcacaaaagc caactggtgt ggatggcgtg ccacagcgcg gcgttcgaag
      481 acctgcgtgt gagcagcttt atccgtggta agaaagttat tccgcgtggc aagctgagca
      541 cccgtggcgt gcagatcgcg agcaacgaga acgttgaaac catggatagc aacaccctgg
      601 agctgcgtag ccgttactgg gcgattcgta cccgtagcgg tggcaacacc aaccagcaaa
      661 aagcgagcgc gggtcagatc agcgtgcaac cgaccttcag cgttcaacgt aacctgccgt
      721 ttgaacgtgc gaccgtgatg gcggcgttca gcggtaacaa cgagggccgt accagcgaca
      781 tgcgtaccga agttattcgt atgatggaga gcgcgaagcc ggaagatctg agcttccagg
      841 gtcgtggcgt gtttgagctg agcgacgaaa aagcgaccaa cccgatcgtt ccgagctttg
      901 atatgagcaa cgaaggtagc tacttctttg gcgacaacgc ggaggaatat gataacaagc
      961 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     1021 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1081 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1141 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1201 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1261 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1321 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1381 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1441 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1501 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1561 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     1621 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1681 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     1741 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     1801 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     1861 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     1921 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     1981 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     2041 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     2101 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     2161 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     2221 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     2281 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     2341 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2401 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     2461 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     2521 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     2581 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2641 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2701 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2761 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2821 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2881 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2941 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3001 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3061 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3121 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3181 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3241 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3301 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3361 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3421 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     3481 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3541 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3601 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3661 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3721 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3781 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3841 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3901 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     3961 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4021 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4081 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4141 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     4201 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4261 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     4321 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4381 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4441 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4501 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4561 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4621 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4681 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4741 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4801 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4861 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4921 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     4981 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5041 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     5101 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     5161 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     5221 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5281 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5341 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5401 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5461 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5521 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5581 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5641 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5701 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5761 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5821 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     5881 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     5941 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     6001 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     6061 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa
     6121 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     6181 ttaactttaa gaaggagata tacatatgca ccatcatcat catcattctt ctggtctggt
     6241 gccacgcggt tctggtatga aagaaaccgc tgctgctaaa ttcgaacgcc agcacatgga
     6301 cagcccagat ctgggtaccg acgacgacga caaggccatg gctgatatcg gatccatggc
     6361 gagccagggt accaagcgta gctacgagca aatggaaacc ggtggcgagc gtcaggacac
     6421 caccgaaatc cgtgcgagcg tgggtcgtat gatcggtggc attggccgtt tctacattca
     6481 aatgtgcacc gagctgaaac tgagcgacta tgatggtcgt ctgatccaga acagcatcac
     6541 cattgagcgt atggttctga gcgcgtttga cgaacgtcgt aacaagtatc tggaggaaca
     6601 cccgagcgcg ggtaaagatc cgaagaaaac cggtggcccg atctaccgtc gtattgacgg
     6661 caagtggacc cgtgagctga tcctgtatga taaagaggaa attcgtcgtg tgtggcgtca
     6721 agcgaacaac ggtgaagacg cgaccgcggg cctgacccac atcatgattt ggcacagcaa
     6781 cctgaacgac gcgacctacc aacgtacccg tgcgctggtt cgtaccggta tggacccgcg
     6841 tatgtgcagc ctgatgcagg gcagcaccct gccgcgtcgt agcggtgcgg cgg
//
