LOCUS       sequence-402810-        6489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses split-intein fused MSP1E3D1 for circularization during 
            expression (external His-Tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6489)
  AUTHORS   Daniilidis M, Brandl MJ, Hagn F
  TITLE     The Advanced Properties of Circularized MSP Nanodiscs Facilitate 
            High-resolution NMR Studies of Membrane Proteins.
  JOURNAL   J Mol Biol. 2022 Dec 30;434(24):167861. doi: 
            10.1016/j.jmb.2022.167861. Epub 2022 Oct 20.
  PUBMED    36273602
REFERENCE   2  (bases 1 to 6489)
  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: "J Mol 
            Biol."; date: "2022-12-30"; volume: "434(24):167861. doi"; pages: " 
            10.1016/j.jmb.2022.167861. Epub 2022 Oct 20"
COMMENT     Sequence Label: pET28a-cMSP1E3D1
FEATURES             Location/Qualifiers
     source          1..6489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1373..1393)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1394..1411)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1430..1452
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1467..1491
                     /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)."
     primer_bind     complement(1491..1510)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1492..1510)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1594..1613)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1819..1896
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1897..2979
                     /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
                     MSGFQQTMQMLNEGIVPTAMLVVNDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1916..1935)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3788..3979
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3973..3995)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4081..4220
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4136..4155
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4235..4252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4406..4994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4486..4505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5105..5124)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5116..5931
                     /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             5116..5931
                     /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     complement(5189..5208)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6023..6478)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6160..6181)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6372..6391
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cattcggcag gttgtcaacg cgcatcagat ccagttcacg ttcgaagatt tcatcaatcg
      241 gcagcatttg accgtccacc gtcataaatt tgtgatcttt ggtggcgcgg atcagagaac
      301 catcttccag acaatattca aacacttcct gttcgccacg gtcatgccat tgcgcgaccg
      361 gctgcgtgta aatgttgcca ttgttgtcaa cgctatacac ggtgcattca atgcgttttt
      421 ccacaatttt accaatcggc agcagaccgt attccaccgt caggatttcc gtttcatagg
      481 acagacaacc ctgggtattc agctttttag tatattcttc cagagctgac agaaaactga
      541 ctttaaagct ctcaagaaca ggcaataagc cctggcgtag atcttccagc gccggtttcg
      601 ctttttcgct caaggtgctc aggtgttctg tcgctttcgc gtggtactca gccaagcggg
      661 cacccccgtt ttctttcagc gcctcgagac gtgcggccaa acgctggcga agttcatccg
      721 aatacggcgc caaatgagtc cggagtgcat caacatgtgc acgggcgcga tcgcgcattt
      781 cttcgcctaa tgggctgagc ttctcttgga gctcatggag tttttggcgt gccccctctt
      841 gcagttccgc acgcagcggt tccacctttt gacgatataa ttccatctct tcctgccatt
      901 ttttctgaaa gtcatcgaga tatggcgcca aatgagtccg gagtgcatca acatgtgcac
      961 gggcgcgatc gcgcatttct tcgcctaatg ggctgagctt ctcttggagc tcatggagtt
     1021 tttggcgtgc cccctcttgc agttccgcac gcagcggttc caccttttga cgatataatt
     1081 ccatctcttc ctgccatttt ttctgaaagt catcgagata tggctgaacc ttggccttca
     1141 cctcttctaa atctttggac atttcctgac gcagtccctc ggtttctttt tccaggttgt
     1201 cccagaattc ctgcgtcacg gggcccagtt gttcgcgaag tttactgaag gtagaaccgt
     1261 taaagcagtt ggatgcgata aagccatttt tcagggcaaa gttgtggtca cgttcgacac
     1321 caatatcgta gacattctgt ttgcccagat atttacgggt cgcgatttta atgccctgaa
     1381 aatacaggtt ttcatgatga tgatgatgat ggctgctgcc catggtatat ctccttctta
     1441 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
     1501 agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg
     1561 gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg
     1621 aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag
     1681 gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg
     1741 cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg
     1801 gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga
     1861 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     1921 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     1981 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     2041 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     2101 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     2161 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     2221 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     2281 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     2341 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     2401 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     2461 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     2521 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     2581 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgt caacgatcag
     2641 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     2701 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     2761 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     2821 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     2881 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2941 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     3001 ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt
     3061 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     3121 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
     3181 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
     3241 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
     3301 tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
     3361 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     3421 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     3481 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     3541 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     3601 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     3661 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     3721 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     3781 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     3841 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     3901 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     3961 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     4021 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     4081 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     4141 tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa
     4201 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4261 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4321 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4381 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4441 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4501 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4561 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4621 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4681 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4741 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4801 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4861 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4921 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4981 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5041 aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt
     5101 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta
     5161 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     5221 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     5281 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     5341 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     5401 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     5461 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     5521 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     5581 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     5641 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     5701 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     5761 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     5821 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     5881 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt
     5941 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6001 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     6061 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     6121 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     6181 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     6241 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     6301 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     6361 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6421 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     6481 cattcgcca
//
