LOCUS       sequence-317653-        5881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mutated membrane scaffold protein (""dark"" 
            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: pD-MSP1D1
FEATURES             Location/Qualifiers
     source          1..5881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..76
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     160..179
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        160..178
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    179..203
                     /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             218..240
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             253..273
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             295..315
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             905..922
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(975..993)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      989..1036
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1073..1528
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1160..1179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1370..1391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1620..2435)
                     /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(1620..2435)
                     /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     2343..2362
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2427..2446
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2557..3145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3046..3065
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3299..3316
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3331..3470
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3396..3415)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3556..3578
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3572..3763)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4572..5654)
                     /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     5616..5635
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5655..5732)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
       61 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
      121 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
      181 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
      241 tataccatgg gtcatcatca tcatcatcat cacgattatg atattcctac tactgagaat
      301 ttgtattttc agggttctac cttcagtaaa cttcgcgaac aactgggccc cgtgacgcag
      361 gaattcttcg acaacctgga aaaagaaacc gagggactgc gtcaggaaat gtccaaagat
      421 ctagaagagg tgaaggccaa ggttcagcca tatctcgatg actttcagaa aaaattccag
      481 gaagagatgg aattatatcg tcaaaaggtg gaaccgctgc gtgcggaact gcaagaggga
      541 gcaagacaaa aactccatga gctccaagag aagctcagcc cattaggcga agaaatgcgc
      601 gatcgcgccc gtgcacatgt tgatgcactc cggactcatt tggcgccgta ttcggatgaa
      661 cttcgccagc gtttggccgc acgtctcgag gcgctgaaag aaaacggggg tgcccgcttg
      721 gctgagtacc acgcgaaagc gacagaacac ctgagcacct tgagcgaaaa agcgaaaccg
      781 gcgctggaag atctacgcca gggcttattg cctgttcttg agagctttaa agtcagtttt
      841 ctgtcagctc tggaagaata tactaaaaag ctgaataccc agtaagcttg cggccgcact
      901 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
      961 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1021 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1081 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1141 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1201 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1261 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1321 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1381 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1441 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     1501 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     1561 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     1621 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     1681 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     1741 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     1801 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     1861 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     1921 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     1981 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     2041 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     2101 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     2161 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2221 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     2281 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     2341 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     2401 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     2461 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2521 agcgtcagac cccgtagaaa agatcaaagg atcttcttga aatccttttt ttctgcgcgt
     2581 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2641 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2701 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2761 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2821 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2881 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2941 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3001 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3061 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3121 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3181 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3241 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3301 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3361 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3421 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3481 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3541 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3601 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     3661 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     3721 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     3781 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     3841 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     3901 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     3961 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4021 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4081 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4141 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4201 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4261 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4321 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4381 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4441 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4501 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4561 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4621 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     4681 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     4741 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     4801 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     4861 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     4921 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     4981 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5041 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5101 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5161 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5221 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5281 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5341 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5401 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5461 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5521 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5581 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     5641 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     5701 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     5761 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     5821 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     5881 a
//
