LOCUS       sequence-317306-        4081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses codon optimized SARS-CoV-2 NSP8 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4081)
  AUTHORS   Dangerfield TL, Huang NZ, Johnson KA
  TITLE     Remdesivir Is Effective in Combating COVID-19 because It Is a Better
            Substrate than ATP for the Viral RNA-Dependent RNA Polymerase.
  JOURNAL   iScience. 2020 Nov 28:101849. doi: 10.1016/j.isci.2020.101849.
  PUBMED    33283177
REFERENCE   2  (bases 1 to 4081)
  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: "iScience. 
            2020 Nov 28:101849. doi: 10.1016/j.isci.2020.101849."
COMMENT     Sequence Label: pcI-ts,ind+-(NSP8)
FEATURES             Location/Qualifiers
     source          1..4081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      634..720
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      812..839
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(933..949)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(933..949)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(946..968)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    957..973
                     /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)."
     promoter        complement(join(981..987,988..1005,1006..1011))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1026..1047
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1164..1181)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1335..1923)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1415..1434)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2094..2885,2886..2954))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2717..2736
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(3270..3983)
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     RBS             4072..4080
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
ORIGIN
        1 catatggcta tagcatctga attttcaagt ctacccagtt atgcggcgtt tgctaccgcc
       61 caagaggcgt acgagcaggc ggttgctaat ggtgattctg aagtcgtgct gaagaaactg
      121 aaaaagtccc tgaacgtggc caaatccgag ttcgatcgtg acgccgctat gcaacgtaaa
      181 ttggaaaaaa tggcggacca ggctatgacc cagatgtaca agcaggcacg ttctgaagat
      241 aagcgcgcga aagttaccag cgcaatgcaa accatgctgt tcacgatgct gcgtaagctg
      301 gacaacgatg cgttgaacaa catcatcaat aatgcaagag acggttgtgt tccgttgaac
      361 attattccgt tgaccaccgc ggcgaaactt atggtcgtga tcccggatta taatacctac
      421 aagaacacgt gcgacggcac cacgtttacc tatgcgagcg cgttatggga aattcaacaa
      481 gttgttgacg cagacagcaa gatcgtgcag ctgagcgaga tcagcatgga taacagcccg
      541 aatctggcat ggccactgat tgtgactgct ctccgcgcga actcggccgt aaaactgcag
      601 taagaattca tgcagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
      661 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
      721 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
      781 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
      841 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat
      901 gagacaatag atctaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      961 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     1021 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1081 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1141 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1201 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1261 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1321 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     1381 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     1441 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1501 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     1561 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1621 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1681 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1741 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     1801 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1861 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1921 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1981 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2041 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2101 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2161 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2221 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2281 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2341 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2401 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2461 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2521 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2581 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2641 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2701 ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2761 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2821 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2881 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2941 tgttgaatac tcatactctt cctttttcaa tattatgtaa gcagacagtt ttattgttca
     3001 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct
     3061 ttgttgaata aatcgaactt ttgctgagtt gactccccgc gcggacatta attgcgttgc
     3121 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3181 aacgcgcggg gagaggcggt ttgcgtattg ggcgccatag acgtctttga attgttatca
     3241 gctatgcgcc gaccagaaca ccttgccgat cagccaaacg tctcttcagg ccactgacta
     3301 gcgataactt tccccacaac ggaacaactc tcattgcatg ggatcattgg gtactgtggg
     3361 tttagtggtt gtaaaaacac ctgaccgcta tccctgatca gtttcttgaa ggtaaactca
     3421 tcacccccaa gtctggctat gcagaaatca cctggctcaa cagcctgctc agggtcaacg
     3481 agaattaaca ttccgtcagg aaagcttggc ttggagcctg ttggtgcggt catggaatta
     3541 ccttcaacct caagccagaa tgcagaatca ctggcttttt tggttgtgct tacccatctc
     3601 tccgcatcac ctttggtaaa ggttctaagc tcaggtgaga acatccctgc ctgaacatga
     3661 gaaaaaacag ggtactcata ctcacttcta agtgacggct gcatactaac cgcttcatac
     3721 atctcgtaga tttctctggc gattgaaggg ctaaattctt caacgctaac tttgagaatt
     3781 tttgtaagca atgcggcgtt ataagcattt aatgcattga tgccattaaa taaagcacca
     3841 acgcctgact gccccatccc catcttgtct gcgacagatt cctgggataa gccaagttca
     3901 tttttctttt tttcataaat tgctttaagg cgacgtgcgt cctcaagctg ctcttgtgtt
     3961 aatggtttct tttttgtgct catacgttaa atctatcacc gcaagggata aatatctaac
     4021 accgtgcgtg ttgactattt tacctctggc ggtgataatg gttgcatgta ctaaggaggt
     4081 t
//
