LOCUS       sequence-267556-        7038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Covalently circularized nanodisc NW30 for studying membrane proteins
            and viral entry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7038)
  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 7038)
  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: NW30
FEATURES             Location/Qualifiers
     source          1..7038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     83..102
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        83..101
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    102..126
                     /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             141..163
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             182..199
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             209..226
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             242..259
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             260..280
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1958..1975
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1985..2002
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2055..2073)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2069..2116
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2153..2608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2240..2259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2450..2471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2700..3515)
                     /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(2700..3515)
                     /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     3423..3442
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3507..3526
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3637..4225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4126..4145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4379..4396
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4411..4550
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4476..4495)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4636..4658
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4652..4843)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5652..6734)
                     /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     6696..6715
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6735..6812)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7018..7037
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
       61 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
      121 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag
      181 ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata tgggcagctc
      241 tcatcaccat caccatcacg aaaacctgta ttttcagggc agtaccttct ccaaactgcg
      301 cgaacagctg ggtccggtga cgcaagaatt ctgggataac ctggaaaaag aaaccgaagg
      361 cctgcgtcag gaaatgagta aagacctgga agaagtgaaa gccaaagttc aaccgtatct
      421 ggatgacttc cagaaaaaat ggcaagaaga aatggaactg taccgccaga aagtggaacc
      481 gctgcgtgcc gaactgcagg aaggcgcacg ccaaaaactg catgaactgc aagaaaaact
      541 gtcaccgctg ggtgaagaaa tgcgtgatcg cgcccgtgca catgttgacg ctctgcgtac
      601 ccacctggcg ccgtattcgg atgaactgcg tcagcgtctg gccgcacgtc tggaagcgct
      661 gaaagaaaac ggcggtgccc gtctggcaga ataccatgct aaagcgaccg aacacctgtc
      721 aacgctgtcg gaaaaagcta aaccggccct ggaagatctg cgccagggtc tgctgccggt
      781 gctggaatca tttaaagtta gcttcctgtc tgcgctggaa gaatatacga aaaaactgaa
      841 cacccagggc acgccggtta cccaggaatt ttgggacaat ctggaaaaag aaacggaagg
      901 cctgcgccag gaaatgtcga aagacctgga agaagtcaaa gccaaagtgc agccgtacct
      961 ggacgatttt cagaaaaaat ggcaagaaga aatggagctg taccgccaga aagtcgaacc
     1021 gctgcgcgcg gaactgcagg aaggcgcccg tcagaaactg catgaactgc aggaaaaact
     1081 gagcccgctg ggcgaagaaa tgcgtgatcg tgcccgcgca catgttgatg ccctgcgcac
     1141 ccacctggca ccgtattctg atgaactgcg ccagcgcctg gcagctcgtc tggaagccct
     1201 gaaagaaaat ggcggtgccc gcctggccga atatcacgcc aaagccaccg aacacctgag
     1261 tacgctgtcc gaaaaagcta aaccggcgct ggaagatctg cgccaaggtc tgctgccggt
     1321 cctggaatca tttaaagtgt cattcctgtc ggcactggaa gaatacacga aaaaactgaa
     1381 cacgcagggc accccggtta cccaggaatt ctgggacaac ctggaaaagg agacggaagg
     1441 cctgcgtcaa gaaatgagca aagatctgga agaagttaaa gcgaaagtcc agccgtacct
     1501 ggatgacttt cagaaaaaat ggcaagaaga aatggagtta taccgccaga aagttgaacc
     1561 gctgcgtgca gaactgcagg aaggcgctcg tcagaaactg cacgagttac aagaaaaact
     1621 gagtccgctg ggagaggaaa tgcgcgaccg tgcccgtgca catgttgatg cactgcgtac
     1681 gcatctggca ccgtattccg acgaactgcg tcaacgcctg gccgcccgcc tggaagcgtt
     1741 aaaggaaaat ggcggtgccc gtctggccga atatcacgcg aaagccacgg aacatctgag
     1801 cacgctgtct gaaaaagcca agcctgccct ggaagatctg cgccaggggc tgctgccggt
     1861 tctggaaagc tttaaagtca gtttcctgtc cgcgctggaa gaatatacca aaaaactgaa
     1921 tacccagctg ccgggtaccg gtgcagctgc cctggaacac catcatcacc atcactaact
     1981 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     2041 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     2101 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     2161 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2221 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2281 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2341 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2401 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2461 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2521 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2581 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     2641 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     2701 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     2761 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     2821 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     2881 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     2941 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     3001 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     3061 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     3121 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     3181 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     3241 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     3301 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     3361 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     3421 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     3481 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     3541 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3601 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3661 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3721 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3781 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3841 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3901 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3961 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4021 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4081 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4141 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4201 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4261 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4321 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4381 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     4441 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4501 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     4561 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4621 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4681 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     4741 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4801 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4861 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4921 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4981 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     5041 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     5101 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     5161 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     5221 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     5281 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     5341 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     5401 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     5461 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     5521 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     5581 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     5641 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5701 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     5761 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5821 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5881 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5941 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     6001 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     6061 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     6121 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     6181 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     6241 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     6301 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     6361 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     6421 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     6481 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     6541 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     6601 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     6661 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     6721 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6781 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6841 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     6901 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     6961 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     7021 gatgtcggcg atataggc
//
