LOCUS       sequence-182625-        5884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanodisc scaffold protein belt. Mutant of MSP1D1 from the Sligar Lab
            to include an additional T 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: pMSP1D1T1
FEATURES             Location/Qualifiers
     source          1..5884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     60..79
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     163..182
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        163..181
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    182..206
                     /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             221..243
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             256..276
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             298..318
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             911..928
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(981..999)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      995..1042
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1079..1534
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1166..1185)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1376..1397
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1626..2441)
                     /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(1626..2441)
                     /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     2349..2368
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2433..2452
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2563..3151
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3052..3071
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3305..3322
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3337..3476
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3402..3421)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3562..3584
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3578..3769)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4578..5660)
                     /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     5622..5641
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5661..5738)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
       61 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
      121 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
      181 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
      241 agatatacca tgggtcatca tcatcatcat catcacgatt atgatattcc tactactgag
      301 aatttgtatt ttcagggttc taccaccttc agtaaacttc gcgaacaact gggccccgtg
      361 acgcaggaat tctgggacaa cctggaaaaa gaaaccgagg gactgcgtca ggaaatgtcc
      421 aaagatttag aagaggtgaa ggccaaggtt cagccatatc tcgatgactt tcagaaaaaa
      481 tggcaggaag agatggaatt atatcgtcaa aaggtggaac cgctgcgtgc ggaactgcaa
      541 gagggggcac gccaaaaact ccatgagctc caagagaagc tcagcccatt aggcgaagaa
      601 atgcgcgatc gcgcccgtgc acatgttgat gcactccgga ctcatttggc gccgtattcg
      661 gatgaacttc gccagcgttt ggccgcacgt ctcgaggcgc tgaaagaaaa cgggggtgcc
      721 cgcttggctg agtaccacgc gaaagcgaca gaacacctga gcaccttgag cgaaaaagcg
      781 aaaccggcgc tggaagatct acgccagggc ttattgcctg ttcttgagag ctttaaagtc
      841 agttttctgt cagctctgga agaatatact aaaaagctga atacccagta agcttgcggc
      901 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
      961 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1021 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     1081 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1141 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1201 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1261 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1321 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1381 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1441 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1501 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     1561 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     1621 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     1681 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     1741 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     1801 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     1861 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     1921 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     1981 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2041 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2101 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2161 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2221 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2281 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2341 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2401 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     2461 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     2521 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgaaatc ctttttttct
     2581 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2641 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2701 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2761 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2821 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2881 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2941 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3001 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3061 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3121 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3181 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3241 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3301 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3361 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3421 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3481 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3541 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3601 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3661 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3721 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3781 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3841 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     3901 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     3961 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4021 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4081 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4141 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4201 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4261 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4321 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4381 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4441 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4501 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4561 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4621 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4681 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4741 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4801 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     4861 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4921 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     4981 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5041 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5101 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5161 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5221 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5281 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5341 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5401 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5461 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5521 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5581 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5641 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5701 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     5761 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     5821 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     5881 cacc
//
