LOCUS       sequence-395160-        5554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of codon optimized SARS-CoV-2 ORF9c tagged with
            an N-terminus His tag followed by a TEV cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5554)
  AUTHORS   Ghelichkhani F, Gonzalez FA, Kapitonova MA, Rozovsky S
  TITLE     Selenoprotein S Interacts with the Replication and Transcription 
            Complex of SARS-CoV-2 by Binding nsp7.
  JOURNAL   J Mol Biol. 2023 Apr 15;435(8):168008. doi: 
            10.1016/j.jmb.2023.168008. Epub 2023 Feb 10.
  PUBMED    36773692
REFERENCE   2  (bases 1 to 5554)
  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: "J Mol 
            Biol."; date: "2023-04-15"; volume: "435(8):168008. doi"; pages: " 
            10.1016/j.jmb.2023.168008. Epub 2023 Feb 10"
COMMENT     Sequence Label: pET-28a(+)-6xHis-TEV-ORF9c
FEATURES             Location/Qualifiers
     source          1..5554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(429..449)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(459..476)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             495..517
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    532..556
                     /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)."
     primer_bind     complement(556..575)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(557..575)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(659..678)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        884..961
                     /gene="lacI"
                     /label=lacI promoter
     CDS             962..2044
                     /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     complement(981..1000)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2853..3044
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3038..3060)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3146..3285
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3201..3220
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3300..3317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3471..4059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3551..3570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4170..4189)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4181..4996
                     /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             4181..4996
                     /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     complement(4254..4273)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5088..5543)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5225..5246)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5437..5456
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccttagtcc gtcaggcagc aacacagcag caacagcatc
      241 accgccattg ccagccactc caagagtaac agttcaccaa ccgccgcttg cagttggatc
      301 tcctgaacgg tggccacgac gtgatgcgga accaacagcg gtttcacagc cgcttctgcg
      361 cccttctggg tgctagcctt ctggcagtgt tgttctttta aaaaattata acatgattgt
      421 agcatatggc cctgaaaata aagattctcg ccgctgctgt gatgatgatg atgatggctg
      481 ctgcccatgg tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt
      541 atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc
      601 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
      661 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
      721 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
      781 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
      841 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
      901 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
      961 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     1021 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     1081 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     1141 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     1201 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     1261 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     1321 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     1381 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     1441 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     1501 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     1561 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     1621 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     1681 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     1741 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     1801 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     1861 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     1921 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     1981 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2041 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     2101 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     2161 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     2221 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     2281 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     2341 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga
     2401 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     2461 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     2521 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     2581 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     2641 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     2701 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     2761 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     2821 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     2881 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     2941 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     3001 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     3061 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     3121 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     3181 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg
     3241 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     3301 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3361 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3421 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3481 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3541 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3601 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3661 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3721 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3781 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3841 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3901 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3961 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4021 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4081 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgaacaat
     4141 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     4201 aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     4261 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc
     4321 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     4381 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     4441 tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt
     4501 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     4561 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     4621 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     4681 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     4741 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     4801 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     4861 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     4921 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     4981 cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt atttagaaaa
     5041 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     5101 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5161 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5221 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5281 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5341 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     5401 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     5461 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     5521 taatgcgccg ctacagggcg cgtcccattc gcca
//
