LOCUS       sequence-340025-        5871 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Covalently circularized nanodisc NW11 for studying membrane proteins
            and viral entry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5871)
  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 5871)
  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: NW11
FEATURES             Location/Qualifiers
     source          1..5871
                     /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             118..138
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             736..753
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(806..824)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      820..867
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      904..1359
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(991..1010)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1201..1222
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1451..2266)
                     /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(1451..2266)
                     /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     2174..2193
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2258..2277
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2388..2976
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2877..2896
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3130..3147
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3162..3301
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3227..3246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3387..3409
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3403..3594)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4403..5485)
                     /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     5447..5466
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5486..5563)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5769..5788
                     /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 tcatcatgaa
      121 aacctgtatt ttcagggcag cacctttagc aaactgcgtg aacagctggg cccggtgacc
      181 caggaatttt gggataacct ggaaaaagaa accgaaggcc tgcgtcagga aatgagcaaa
      241 gatctggaag aggtgaaagc gaaagtgcag ccgtatctgg atgactttca gaaaaaatgg
      301 caggaagaga tggaactgta tcgtcagaaa gtggaaccgc tgcgtgcgga actgcaggaa
      361 ggcgcgcgtc agaaactgca tgaactgcag gaaaaactga gcccgctggg cgaagagatg
      421 cgtgatcgtg cgcgtgcgca tgtggatgcg ctgcgtaccc atctggcgcc gtatagcgat
      481 gaactgcgtc agcgtctggc ggcccgtctg gaagcgctga aagaaaacgg cggtgcgcgt
      541 ctggcggaat atcatgcgaa agcgaccgaa catctgagca ccctgagcga aaaagcgaaa
      601 ccggcgctgg aagatctgcg tcagggcctg ctgccggtgc tggaaagctt taaagtgagc
      661 tttctgagcg cgctggaaga gtataccaaa aaactgaaca cccagctgcc gggcaccggc
      721 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
      781 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      841 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
      901 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
      961 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1021 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1081 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1141 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1201 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1261 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1321 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1381 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1441 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1501 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1561 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1621 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1681 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     1741 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     1801 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     1861 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     1921 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     1981 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2041 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2101 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2161 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2221 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2281 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2341 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2401 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2461 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2521 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2581 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2641 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2701 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2761 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2821 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2881 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2941 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3001 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3061 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3121 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3181 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3241 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3301 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3361 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3421 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3481 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3541 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3601 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3661 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     3721 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     3781 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     3841 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     3901 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     3961 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4021 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4081 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4141 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4201 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4261 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4321 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4381 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4441 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4501 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4561 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4621 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4681 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     4741 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     4801 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     4861 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     4921 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     4981 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5041 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5101 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5161 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5221 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5281 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5341 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5401 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5461 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5521 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5581 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5641 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5701 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     5761 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     5821 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
