LOCUS       sequence-318420-        6016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ""extended"" membrane scaffold protein 
            MSP1E2D1 for Nanodisc formation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6016)
  AUTHORS   Ritchie TK, Grinkova YV, Bayburt TH, Denisov IG, Zolnerciks JK, 
            Atkins WM, Sligar SG
  TITLE     Chapter 11 - Reconstitution of membrane proteins in phospholipid 
            bilayer nanodiscs.
  JOURNAL   Methods Enzymol. 2009;464:211-31. doi: 
            10.1016/S0076-6879(09)64011-8.
  PUBMED    19903557
REFERENCE   2  (bases 1 to 6016)
  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: "Methods 
            Enzymol."; date: "2009"; volume: "464:211-31. doi"; pages: " 
            10.1016/S0076-6879(09)64011-8"
COMMENT     Sequence Label: pMSP1E2D1
FEATURES             Location/Qualifiers
     source          1..6016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     130..149
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        130..148
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    149..173
                     /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             188..210
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             223..243
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             265..285
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1010..1027
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1080..1098)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1094..1141
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1178..1633
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1265..1284)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1475..1496
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1725..2540)
                     /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(1725..2540)
                     /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     2448..2467
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2532..2551
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2662..3250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3151..3170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3404..3421
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3436..3575
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3501..3520)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3661..3683
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3677..3868)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4677..5759)
                     /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     5721..5740
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5760..5837)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
       61 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      121 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
      181 aaataatttt gtttaacttt aagaaggaga tataccatgg gtcatcatca tcatcatcat
      241 cacgattatg atattcctac tactgagaat ttgtattttc agggttctac cttcagtaaa
      301 cttcgcgaac aactgggccc cgtgacgcag gaattctggg acaacctgga aaaagaaacc
      361 gagggactgc gtcaggaaat gtccaaagat ttagaagagg tgaaggccaa ggttcagcca
      421 tatctcgatg actttcagaa aaaatggcag gaagagatgg aattatatcg tcaaaaggtg
      481 gaaccgctgc gtgcggaact gcaagagggg gcacgccaaa aactccatga gctccaagag
      541 aagctcagcc catatctcga tgactttcag aaaaaatggc aggaagagat ggaattatat
      601 cgtcaaaagg tggaaccgct gcgtgcggaa ctgcaagagg gggcacgcca aaaactccat
      661 gagctccaag agaagctcag cccattaggc gaagaaatgc gcgatcgcgc ccgtgcacat
      721 gttgatgcac tccggactca tttggcgccg tattcggatg aacttcgcca gcgtttggcc
      781 gcacgtctcg aggcgctgaa agaaaacggg ggtgcccgct tggctgagta ccacgcgaaa
      841 gcgacagaac acctgagcac cttgagcgaa aaagcgaaac cggcgctgga agatctacgc
      901 cagggcttat tgcctgttct tgagagcttt aaagtcagtt ttctgtcagc tctggaagaa
      961 tatactaaaa agctgaatac ccagtaataa gcttgcggcc gcactcgagc accaccacca
     1021 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1081 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1141 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1201 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1261 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1321 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1381 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1441 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1501 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1561 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1621 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1681 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     1741 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     1801 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     1861 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1921 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1981 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     2041 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     2101 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2161 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2221 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2281 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2341 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2401 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2461 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     2521 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     2581 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2641 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2701 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2761 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2821 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2881 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2941 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3001 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3061 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3121 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3181 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3241 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3301 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3361 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3421 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3481 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3541 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3601 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3661 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3721 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3781 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3841 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3901 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3961 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4021 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4081 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4141 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4201 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4261 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4321 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4381 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4441 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4501 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4561 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4621 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4681 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4741 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4801 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4861 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4921 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4981 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5041 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5101 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5161 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5221 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5281 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5341 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5401 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5461 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5521 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5581 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5641 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5701 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5761 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5821 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5881 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5941 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     6001 agcgctcatg agcccg
//
