LOCUS       sequence-317666-        5881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mutated membrane scaffold protein 
            (""ultra-bright"" MSP1D1) for Nanodisc formation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5881)
  AUTHORS   McLean MA, Denisov IG, Grinkova YV, Sligar SG
  TITLE     Dark, Ultra-Dark and Ultra-Bright Nanodiscs for membrane protein 
            investigations.
  JOURNAL   Anal Biochem. 2020 Oct 15;607:113860. doi: 10.1016/j.ab.2020.113860.
            Epub 2020 Aug 1.
  PUBMED    32750355
REFERENCE   2  (bases 1 to 5881)
  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"; journalName: "Anal 
            Biochem."; date: "2020-10-15"; volume: "607:113860. doi"; pages: " 
            10.1016/j.ab.2020.113860. Epub 2020 Aug 1"
COMMENT     Sequence Label: pUB-MSP1D1
FEATURES             Location/Qualifiers
     source          1..5881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     145..164
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        145..163
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    164..188
                     /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             203..225
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             238..258
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             280..300
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             890..907
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(960..978)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      974..1021
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1058..1513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1145..1164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1355..1376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1605..2420)
                     /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(1605..2420)
                     /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     2328..2347
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2412..2431
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2542..3130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3031..3050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3284..3301
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3316..3455
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3381..3400)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3541..3563
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3557..3748)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4557..5639)
                     /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     5601..5620
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5640..5717)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
       61 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
      121 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      181 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catgggtcat
      241 catcatcatc atcatcacga ttatgatatt cctactactg agaatttgta ttttcagggt
      301 tctaccttca gtaaacttcg cgaacaactg ggccccgtga cgcaggaatt ctgggacaac
      361 ctggaaaaag aaaccgaggg actgcgtcag gaaatgtcca aagatttaga agaggtgaag
      421 gccaaggttc agccatatct cgatgacttt cagaaaaaat ggcaggaaga gatggaatta
      481 tatcgtcaaa aggtggaacc gctgcgtgcg gaactgcaag agggggcacg ccaaaaactc
      541 catgagctct gggagaagct cagcccatta ggcgaagaaa tgcgcgatcg cgcccgtgca
      601 catgttgatg cactccggac tcatttggcg ccgtattcgg atgaacttcg ccagcgtttg
      661 gccgcacgtc tcgaggcgtg gaaagaaaac gggggtgccc gcttggctga gtaccacgcg
      721 aaagcgacag aacacctgag caccttgagc gaaaaagcga aaccggcgct ggaagatcta
      781 cgccagggct tattgcctgt tcttgagagc tttaaagtca gttttctgtc agctctggaa
      841 gaatggacta aaaagctgaa tacccagtaa gcttgcggcc gcactcgagc accaccacca
      901 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      961 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1021 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1081 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1141 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1201 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1261 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1321 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1381 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1441 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1501 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1561 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     1621 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     1681 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     1741 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1801 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1861 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     1921 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     1981 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2041 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2101 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2161 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2221 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2281 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2341 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     2401 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     2461 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2521 agaaaagatc aaaggatctt cttgaaatcc tttttttctg cgcgtaatct gctgcttgca
     2581 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2641 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2701 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2761 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2821 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2881 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2941 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3001 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3061 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3121 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3181 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3241 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3301 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3361 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3421 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3481 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3541 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3601 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3661 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3721 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3781 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3841 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     3901 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     3961 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4021 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4081 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4141 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4201 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4261 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4321 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4381 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4441 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4501 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4561 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4621 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4681 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4741 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4801 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4861 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4921 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4981 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5041 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5101 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5161 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5221 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5281 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5341 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5401 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5461 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5521 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5581 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5641 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5701 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5761 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5821 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     5881 a
//
