LOCUS       sequence-339788-        6573 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ThuS expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6573)
  AUTHORS   Fujinami D, Garcia de Gonzalo CV, Biswas S, Hao Y, Wang H, Garg N, 
            Lukk T, Nair SK, van der Donk WA
  TITLE     Structural and mechanistic investigations of protein 
            S-glycosyltransferases.
  JOURNAL   Cell Chem Biol. 2021 Dec 16;28(12):1740-1749.e6. doi: 
            10.1016/j.chembiol.2021.06.009. Epub 2021 Jul 21.
  PUBMED    34283964
REFERENCE   2  (bases 1 to 6573)
  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"; doi: 
            "10.1016/j.chembiol.2021.06.009"; journalName: "Cell Chem Biol"; 
            date: "2021-12-16- 16"; volume: "28"; issue: "12"; pages: 
            "1740-1749.e6"
COMMENT     Sequence Label: pThuS
FEATURES             Location/Qualifiers
     source          1..6573
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1438..1455
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1508..1526)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1522..1569
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1606..2061
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1693..1712)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1903..1924
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2153..2968)
                     /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(2153..2968)
                     /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     2876..2895
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2960..2979
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3090..3678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3579..3598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3832..3849
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3864..4003
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3929..3948)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4089..4111
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4105..4296)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5105..6187)
                     /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     6149..6168
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6188..6265)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6471..6490
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcgtgg aaacattaaa cgacttagta
      181 actagactag aacattcgca tccaaattct tctttattaa aagatttaag tttaatacaa
      241 ggaaatgaac aatataacta cattaagtgg ggagatttaa gcaatagtca aaatttaaat
      301 gaactagttt ttcaatatga aaaggcaccg tatccttcaa ttacttgtgg aatacttact
      361 tataatgaag agagatgtat taaaagatgc ttagatagcc ttggaagtca atttgatgaa
      421 attcttgtat tggattctca ttccacagat aatactacaa agatcattaa ccgagatttt
      481 ccaatggtta aagttatata tgagccttgg attgatgatt tttcctttca cagaaataaa
      541 cttataagtt taacaagttc agagtggatt tattatatag atgctgataa ctattgcgtc
      601 gatagtacga ataaatttaa aagagtagcc aaattgattc aatttctttc tatagattgt
      661 ataattagtc cgatgatcaa agaacatatt ggccatgtat atactgacaa tagaaaaatg
      721 ttttctgtaa aaaaagggat tcaattcaaa ggaaaagttc atgaagagcc tataaatgct
      781 gatggtagta ttccacaaaa tattacagtg gatatcatga tttgccatga tgggtatgat
      841 cctgaagtaa ttaatctgtc cgaaaaaaat gatagaaata taaaactaac aagacaaatg
      901 atggaagaag aaccctctaa tccaaaatgg ttatattttt atgctagaga attacattat
      961 gcgagtgaag acacgcatat aattgaaaca cttttaataa aagcaattga tttatataaa
     1021 caaagtacat ataagagata tcaacctgaa gcaatattat tattatgtag tattttattt
     1081 caaaagagac aaatccgaaa gttaaatgaa taccttgatt tattagaaga acttcagcca
     1141 ctttgttctg atgtgaacta ctataggtca cttattttat tctatgatat tcgattaaaa
     1201 acaggaaagc tgttagatac gctaaaatcc agtgaactcg aaaataacaa atatagtttt
     1261 attgattcat caaaagatca cattaaagca ttgttaatag agttatattg ttctatagat
     1321 gactgggaag gcgcatttac gttgtttgat gaacttcaat caacagaggc tagaaataaa
     1381 tttttacgta gagtcaaaac aataaatact catatcagca aaaaaatata actcgagcac
     1441 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1501 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1561 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1621 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1681 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1741 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1801 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1861 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1921 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1981 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     2041 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2101 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     2161 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     2221 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     2281 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2341 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2401 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2461 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2521 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2581 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2641 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2701 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2761 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2821 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2881 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2941 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     3001 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3061 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3121 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3181 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3241 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3301 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3361 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3421 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3481 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3541 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3601 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3661 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3721 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3781 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3841 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3901 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3961 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4021 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4081 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4141 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4201 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4261 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4321 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4381 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4441 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4501 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4561 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4621 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4681 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4741 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4801 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4861 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4921 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4981 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5041 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5101 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5161 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5221 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5281 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5341 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5401 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5461 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5521 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5581 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5641 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5701 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5761 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5821 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5881 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5941 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6001 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6061 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6121 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6181 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6241 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6301 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6361 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6421 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6481 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6541 gcgtagagga tcgagatctc gatcccgcga aat
//
