LOCUS       sequence-324329-        5765 bp    DNA     circular SYN 14-APR-2025
DEFINITION  protein expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5765)
  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 5765)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-ORF8
FEATURES             Location/Qualifiers
     source          1..5765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             48..65
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             75..92
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             99..143
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             159..173
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             582..599
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(652..670)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      666..713
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      750..1205
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(837..856)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1047..1068
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1297..2112)
                     /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(1297..2112)
                     /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     2020..2039
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2104..2123
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2234..2822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2723..2742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2976..2993
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3008..3147
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3073..3092)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3233..3255
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3249..3440)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4249..5331)
                     /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     5293..5312
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5332..5409)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5615..5634
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5722..5741
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5722..5740
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5741..5765
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat acatatgcac catcatcatc
       61 atcattcttc tggtctggtg ccacgcggtt ctggtatgaa agaaaccgct gctgctaaat
      121 tcgaacgcca gcacatggac agcccagatc tgggtaccga cgacgacgac aaggccatgg
      181 ctgatatcgg atccatgaag ttcctggtgt ttctgggtat cattaccacc gttgcggcgt
      241 tccaccagga gtgcagcctg caaagctgca cccagcacca accgtacgtg gttgacgatc
      301 cgtgcccgat ccacttttac agcaagtggt atattcgtgt gggtgcgcgt aaaagcgcgc
      361 cgctgatcga gctgtgcgtt gatgaagcgg gtagcaagag cccgattcag tacatcgaca
      421 ttggcaacta taccgtgagc tgcctgccgt tcaccatcaa ctgccaagaa ccgaaactgg
      481 gtagcctggt ggttcgttgc agcttctacg aggactttct ggaatatcac gatgttcgtg
      541 tggttctgga ctttatttaa aagcttgcgg ccgcactcga gcaccaccac caccaccact
      601 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
      661 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
      721 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
      781 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
      841 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
      901 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
      961 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1021 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1081 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1141 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     1201 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1261 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     1321 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     1381 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     1441 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     1501 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     1561 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     1621 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     1681 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     1741 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     1801 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     1861 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     1921 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     1981 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     2041 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     2101 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     2161 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2221 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2281 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2341 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2401 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2461 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2521 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2581 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2641 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2701 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2761 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2821 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2881 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2941 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3001 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     3061 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3121 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3181 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3241 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     3301 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     3361 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     3421 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     3481 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     3541 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     3601 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     3661 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     3721 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     3781 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     3841 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     3901 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     3961 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     4021 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     4081 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     4141 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     4201 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     4261 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4321 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     4381 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     4441 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     4501 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     4561 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     4621 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     4681 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     4741 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     4801 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     4861 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     4921 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     4981 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     5041 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     5101 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     5161 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     5221 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     5281 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     5341 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     5401 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     5461 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     5521 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     5581 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     5641 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     5701 tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     5761 attcc
//
