LOCUS       sequence-182662-        5884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanodisc scaffold protein belt. Mutant of MSP1D1 from the Sligar Lab
            to include an additional R residue shortly after the TEV cleavage 
            site. Additional mass used as marker in mass spectrometry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5884)
  AUTHORS   Reid DJ, Keener JE, Wheeler AP, Zambrano DE, Diesing JM, 
            Reinhardt-Szyba M, Makarov A, Marty MT
  TITLE     Engineering Nanodisc Scaffold Proteins for Native Mass Spectrometry.
  JOURNAL   Anal Chem. 2017 Nov 7;89(21):11189-11192. doi: 
            10.1021/acs.analchem.7b03569. Epub 2017 Oct 23.
  PUBMED    29048874
REFERENCE   2  (bases 1 to 5884)
  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: 
            "10.1021/acs.analchem.7b03569"; journalName: "Anal Chem"; date: 
            "2017-11-7- 7"; volume: "89"; issue: "21"; pages: "11189-11192"
COMMENT     Sequence Label: pMSP1D1R1
FEATURES             Location/Qualifiers
     source          1..5884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     317..336
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(356..433)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     639..658
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     742..761
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        742..760
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    761..785
                     /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             800..822
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             835..855
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             877..897
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1490..1507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1560..1578)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1574..1621
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1658..2113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1745..1764)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1955..1976
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2205..3020)
                     /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(2205..3020)
                     /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     2928..2947
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3012..3031
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3142..3730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3631..3650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3884..3901
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3916..4055
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3981..4000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4141..4163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4157..4348)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5157..5884,1..355))
                     /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"
ORIGIN
        1 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
       61 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
      121 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
      181 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
      241 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
      301 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
      361 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
      421 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
      481 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
      541 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
      601 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
      661 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
      721 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
      781 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggtcatcat
      841 catcatcatc atcacgatta tgatattcct actactgaga atttgtattt tcagggttct
      901 acccgcttca gtaaacttcg cgaacaactg ggccccgtga cgcaggaatt ctgggacaac
      961 ctggaaaaag aaaccgaggg actgcgtcag gaaatgtcca aagatttaga agaggtgaag
     1021 gccaaggttc agccatatct cgatgacttt cagaaaaaat ggcaggaaga gatggaatta
     1081 tatcgtcaaa aggtggaacc gctgcgtgcg gaactgcaag agggggcacg ccaaaaactc
     1141 catgagctcc aagagaagct cagcccatta ggcgaagaaa tgcgcgatcg cgcccgtgca
     1201 catgttgatg cactccggac tcatttggcg ccgtattcgg atgaacttcg ccagcgtttg
     1261 gccgcacgtc tcgaggcgct gaaagaaaac gggggtgccc gcttggctga gtaccacgcg
     1321 aaagcgacag aacacctgag caccttgagc gaaaaagcga aaccggcgct ggaagatcta
     1381 cgccagggct tattgcctgt tcttgagagc tttaaagtca gttttctgtc agctctggaa
     1441 gaatatacta aaaagctgaa tacccagtaa gcttgcggcc gcactcgagc accaccacca
     1501 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1561 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1621 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1681 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1741 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1801 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1861 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1921 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1981 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2041 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2101 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2161 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     2221 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2281 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2341 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     2401 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     2461 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     2521 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     2581 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2641 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2701 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2761 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2821 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2881 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2941 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     3001 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     3061 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3121 agaaaagatc aaaggatctt cttgaaatcc tttttttctg cgcgtaatct gctgcttgca
     3181 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3241 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3301 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3361 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3421 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3481 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3541 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3601 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3661 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3721 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3781 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3841 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3901 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3961 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4021 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4081 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4141 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4201 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4261 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4321 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4381 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4441 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4501 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4561 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4621 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4681 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4741 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4801 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4861 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4921 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4981 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     5041 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5101 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5161 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5221 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5281 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5341 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5401 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     5461 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5521 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5581 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5641 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5701 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5761 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5821 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5881 ctga
//
