LOCUS       sequence-267558-        8118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Covalently circularized nanodisc NW50 for studying membrane proteins
            and viral entry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8118)
  AUTHORS   Nasr ML, Baptista D, Strauss M, Sun ZJ, Grigoriu S, Huser S, 
            Pluckthun A, Hagn F, Walz T, Hogle JM, Wagner G
  TITLE     Covalently circularized nanodiscs for studying membrane proteins and
            viral entry.
  JOURNAL   Nat Methods. 2017 Jan;14(1):49-52. doi: 10.1038/nmeth.4079. Epub 
            2016 Nov 21.
  PUBMED    27869813
REFERENCE   2  (bases 1 to 8118)
  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: 
            "10.1038/nmeth.4079"; journalName: "Nat Methods"; date: "2017-01"; 
            volume: "14"; issue: "1"; pages: "49-52"
COMMENT     Sequence Label: NW50
FEATURES             Location/Qualifiers
     source          1..8118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        48..66
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    67..91
                     /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             106..128
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             147..164
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             174..191
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             207..224
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             225..245
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3003..3020
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3030..3047
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3100..3118)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3114..3161
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3198..3653
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3285..3304)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3495..3516
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3745..4560)
                     /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(3745..4560)
                     /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     4468..4487
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4552..4571
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4682..5270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5171..5190
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5424..5441
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5456..5595
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5521..5540)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5681..5703
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5697..5888)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6697..7779)
                     /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     7741..7760
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7780..7857)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8063..8082
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
       61 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
      121 gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc ggcctggtgc
      181 cgcgcggcag ccatatgggc agctctcatc accatcacca tcacgaaaac ctgtattttc
      241 agggcagtac cttctccaaa ctgcgcgaac agctgggtcc ggtgacgcaa gaattctggg
      301 ataacctgga aaaagaaacc gaaggcctgc gtcaggaaat gagtaaagac ctggaagaag
      361 tgaaagccaa agttcaaccg tatctggatg acttccagaa aaaatggcaa gaagaaatgg
      421 aactgtaccg ccagaaagtc gaaccgctgc gtgccgaact gcaggaaggc gcacgccaaa
      481 aactgcatga actgcaagaa aaactgagcc cgctgggtga agaaatgcgt gatcgcgccc
      541 gtgcacatgt tgatgctctg cgtacccacc tggccccgta ttctgatgaa ctgcgtcagc
      601 gtctggccgc acgtctggaa gcgctgaaag aaaacggcgg tgcccgtctg gcagaatacc
      661 atgctaaagc gaccgaacac ctgtcaacgc tgtcggaaaa agctaaaccg gccctggaag
      721 atctgcgcca gggtctgctg ccggttctgg aatcatttaa agtcagcttc ctgtctgccc
      781 tggaagaata tacgaaaaaa ctgaacaccc agggcacgcc ggttacccag gaattttggg
      841 acaatctgga aaaagaaacg gaaggcctgc gccaggaaat gtcgaaagac ctggaagaag
      901 tcaaagcaaa agtgcagccg tacctggacg attttcagaa aaaatggcaa gaagaaatgg
      961 agctgtaccg ccagaaagtt gaaccgctgc gcgcggaact gcaggaaggc gcccgtcaga
     1021 aactgcacga actgcaggaa aaactgagtc cgctgggcga agaaatgcgt gatcgtgccc
     1081 gcgcacacgt tgatgccctg cgcacgcacc tggccccgta ttccgatgaa ctgcgccagc
     1141 gcctggcagc tcgtctggaa gccctgaaag aaaatggcgg tgcccgcctg gccgaatatc
     1201 acgccaaagc caccgaacac ctgagtacgc tgtccgaaaa agctaaaccg gcgctggaag
     1261 atctgcgcca aggtctgctg ccggtgctgg aaagctttaa agtttcattc ctgtcggcgc
     1321 tggaagaata cacgaaaaaa ctgaacacgc agggcacccc ggttacccag gaattctggg
     1381 acaacctgga aaaggagacg gaaggcctgc gtcaagaaat gagcaaagat ctggaagaag
     1441 ttaaagccaa agtccagccg tacctggacg actttcagaa aaaatggcaa gaagaaatgg
     1501 agttataccg ccagaaagtg gaaccgctgc gcgctgaact gcaggaaggc gcgcgtcaga
     1561 aactgcatga gttacaggaa aaactgtcac cgctgggaga ggaaatgcgc gaccgtgccc
     1621 gtgcacacgt tgacgccctg cgcacccacc tggcgccgta ttcggacgaa ctgcgtcaac
     1681 gcctggccgc ccgcctggaa gccttaaagg aaaatggcgg tgcccgtctg gccgaatatc
     1741 acgcgaaagc cacggaacat ctgagcacgc tgtctgaaaa agccaagcct gccctggaag
     1801 atctgcgcca ggggctgctg ccggtcctgg aaagttttaa agttagtttc ctgtccgcac
     1861 tggaagagta tacgaaaaaa ctgaataccc agggcacgcc ggtgacccag gaattttggg
     1921 ataatctgga aaaggagacc gaaggcctgc gccaagaaat gtccaaggat ctggaagaag
     1981 tgaaagcgaa agtgcagccg tatctggacg atttccagaa aaaatggcaa gaagaaatgg
     2041 agctatatcg tcaaaaagtt gaaccgctgc gtgcggagtt acaggaaggc gcccgtcaaa
     2101 aactgcacga gttacaagag aagctgagcc cgctgggcga ggagatgcgc gatcgtgccc
     2161 gcgcacatgt tgacgccctg cgtacgcacc tggcaccgta cagcgacgaa ctgcgccaac
     2221 gcctggccgc ccgactggaa gccttaaaag agaatggcgg tgcccgatta gccgaatatc
     2281 acgcaaaagc tacggaacat ctgtctaccc tgagcgaaaa agccaagccc gccctggaag
     2341 atctgcgcca gggcttatta ccggtcctgg aaagcttcaa agttagcttc ctgagcgcgc
     2401 tggaagaata taccaaaaaa ctgaataccc aaggcacccc ggttacccaa gaattttggg
     2461 acaacctgga aaaagagacg gagggcctgc gccaagaaat gagcaaggac ctggaagaag
     2521 tcaaggcgaa agtgcaaccg tacctggacg acttccagaa aaaatggcaa gaagaaatgg
     2581 agctttatcg tcaaaaagtc gaaccgctgc gcgccgagtt acaggaaggc gcgcgtcaaa
     2641 aactgcacga attacaagag aaactgagtc cgctgggaga ggagatgcgc gaccgcgccc
     2701 gtgcacacgt ggacgcactg cgtacgcatc tggcgccgta cagcgatgag ttacgtcaac
     2761 gcctggccgc gcgcctggaa gctttaaagg agaacggcgg tgcccgcctg gcggaatatc
     2821 acgccaaagc tacggaacac ctgagcaccc tgagcgaaaa agcgaagcca gccctggaag
     2881 atctgcgcca gggattattg ccggtcctgg aatctttcaa agtgtcattc ctgagcgccc
     2941 tggaagaata caccaaaaaa ctgaacaccc aactgccggg taccggtgca gctgccctgg
     3001 aacaccatca tcaccatcac taactcgagc accaccacca ccaccactga gatccggctg
     3061 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     3121 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     3181 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3241 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3301 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3361 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3421 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3481 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3541 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3601 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     3661 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3721 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     3781 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     3841 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     3901 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     3961 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     4021 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     4081 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     4141 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     4201 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     4261 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     4321 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     4381 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     4441 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     4501 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     4561 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     4621 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4681 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4741 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4801 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4861 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4921 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4981 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5041 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5101 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5161 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5221 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5281 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5341 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5401 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     5461 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     5521 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5581 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5641 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5701 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5761 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5821 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5881 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5941 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     6001 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     6061 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     6121 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     6181 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     6241 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     6301 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     6361 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     6421 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     6481 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     6541 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     6601 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     6661 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6721 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6781 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     6841 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     6901 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     6961 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     7021 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     7081 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     7141 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     7201 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     7261 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     7321 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     7381 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     7441 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     7501 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     7561 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     7621 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     7681 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     7741 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     7801 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     7861 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     7921 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     7981 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     8041 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     8101 tggcgccggt gatgccgg
//
