LOCUS       sequence-182618-        5887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanodisc scaffold protein belt. Mutant of MSP1D1 from the Sligar Lab
            to include two additional T residues 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 5887)
  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 5887)
  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: pMSP1D1T2
FEATURES             Location/Qualifiers
     source          1..5887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /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             1046..1068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1081..1101
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             1123..1143
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1739..1756
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1809..1827)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1823..1870
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1907..2362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1994..2013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2204..2225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2454..3269)
                     /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(2454..3269)
                     /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     3177..3196
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3261..3280
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3391..3979
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3880..3899
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4133..4150
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4165..4304
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4230..4249)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4390..4412
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4406..4597)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5406..5887,1..601))
                     /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 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggt
     1081 catcatcatc atcatcatca cgattatgat attcctacta ctgagaattt gtattttcag
     1141 ggttctacca ccaccttcag taaacttcgc gaacaactgg gccccgtgac gcaggaattc
     1201 tgggacaacc tggaaaaaga aaccgaggga ctgcgtcagg aaatgtccaa agatttagaa
     1261 gaggtgaagg ccaaggttca gccatatctc gatgactttc agaaaaaatg gcaggaagag
     1321 atggaattat atcgtcaaaa ggtggaaccg ctgcgtgcgg aactgcaaga gggggcacgc
     1381 caaaaactcc atgagctcca agagaagctc agcccattag gcgaagaaat gcgcgatcgc
     1441 gcccgtgcac atgttgatgc actccggact catttggcgc cgtattcgga tgaacttcgc
     1501 cagcgtttgg ccgcacgtct cgaggcgctg aaagaaaacg ggggtgcccg cttggctgag
     1561 taccacgcga aagcgacaga acacctgagc accttgagcg aaaaagcgaa accggcgctg
     1621 gaagatctac gccagggctt attgcctgtt cttgagagct ttaaagtcag ttttctgtca
     1681 gctctggaag aatatactaa aaagctgaat acccagtaag cttgcggccg cactcgagca
     1741 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     1801 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1861 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1921 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1981 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2041 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2101 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2161 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2221 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2281 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     2341 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2401 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     2461 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     2521 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     2581 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     2641 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     2701 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     2761 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     2821 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     2881 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     2941 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     3001 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     3061 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     3121 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     3181 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     3241 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     3301 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3361 agaccccgta gaaaagatca aaggatcttc ttgaaatcct ttttttctgc gcgtaatctg
     3421 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3481 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3541 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3601 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3661 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3721 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3781 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3841 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3901 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3961 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4021 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4081 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4141 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4201 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4261 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4321 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4381 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4441 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4501 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4561 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4621 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4681 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4741 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4801 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4861 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4921 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4981 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5041 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     5101 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5161 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     5221 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     5281 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     5341 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     5401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     5521 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     5581 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     5641 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     5701 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     5761 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     5821 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     5881 ttatgcc
//
