LOCUS       sequence-269584-        5832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Covalently circularized nanodisc NW9 for studying membrane proteins 
            and viral entry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5832)
  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 5832)
  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: NW9
FEATURES             Location/Qualifiers
     source          1..5832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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             88..110
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             129..146
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             147..167
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             699..716
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             726..743
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(796..814)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      810..857
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      894..1349
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(981..1000)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1191..1212
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1441..2256)
                     /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(1441..2256)
                     /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     2164..2183
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2248..2267
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2378..2966
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2867..2886
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3120..3137
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3152..3291
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3217..3236)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3377..3399
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3393..3584)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4393..5475)
                     /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     5437..5456
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5476..5553)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5759..5778
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
       61 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
      121 gttcaagcca ccaccaccac caccacgaaa atctgtactt tcaaggcagc acgttcagca
      181 aactgcgcga acaactgggt ccggttaccc aggaattttg ggataacctg gaaaaagaaa
      241 cggaaggcct gcgccaggaa atgagcaaag atctggaaga agtgaaagcc aaagttcaac
      301 cgtatctgga tgacttccag aaaaaatggc aagaagaaat ggaactgtac cgccagaaag
      361 ttgaaccgct gggtgaagaa atgcgtgatc gcgcacgtgc tcatgttgat gccctgcgta
      421 cccacctggc accgtattcg gatgaactgc gtcagcgtct ggccgcacgt ctggaagcac
      481 tgaaagaaaa tggcggtgca cgtctggctg aataccatgc gaaagccacc gaacacctga
      541 gcacgctgtc tgaaaaagcg aaaccggccc tggaagacct gcgtcagggt ctgctgccgg
      601 tcctggaaag ttttaaagtt agtttcctgt ccgcgctgga agaatatacg aaaaaactga
      661 acacgcaact gccgggtacg ggcgccgctg cactggaaca tcatcatcat catcactaac
      721 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
      781 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      841 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      901 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      961 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1021 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1081 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1141 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1201 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1261 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1321 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     1381 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     1441 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1501 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1561 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     1621 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     1681 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     1741 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1801 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1861 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1921 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     1981 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2041 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2101 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2161 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2221 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     2281 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     2341 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2401 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2461 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2521 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2581 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2641 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2701 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2761 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2821 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2881 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2941 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3001 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3061 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3121 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3181 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3241 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3301 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3361 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3421 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3481 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3541 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3601 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3661 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3721 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3781 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3841 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3901 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3961 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4021 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4081 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4141 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4201 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4261 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4321 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4381 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4441 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4501 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4561 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4621 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     4681 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     4741 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     4801 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4861 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4921 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4981 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5041 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5101 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5161 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5221 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5281 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5341 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5401 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5461 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5521 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5581 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5641 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5701 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     5761 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     5821 tgcgtccggc gt
//
