LOCUS       sequence-404923-        7308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses split-intein fused MSP26 for circularization during 
            expression (internal His-Tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7308)
  AUTHORS   Miehling J, Goricanec D, Hagn F
  TITLE     A Split-Intein-Based Method for the Efficient Production of 
            Circularized Nanodiscs for Structural Studies of Membrane Proteins.
  JOURNAL   Chembiochem. 2018 Sep 17;19(18):1927-1933. doi: 
            10.1002/cbic.201800345. Epub 2018 Jul 30.
  PUBMED    29947468
REFERENCE   2  (bases 1 to 7308)
  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: 
            "10.1002/cbic.201800345"; journalName: "Chembiochem"; date: 
            "2018-09-17- 17"; volume: "19"; issue: "18"; pages: "1927-1933"
COMMENT     Sequence Label: pET28a-cMSP26
FEATURES             Location/Qualifiers
     source          1..7308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     955..974
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(994..1071)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1277..1296
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1380..1399
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1380..1398
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1399..1423
                     /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             1438..1460
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1596..1613
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3622..3640)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3636..3683
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3720..4175
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3807..3826)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4017..4038
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4267..5082)
                     /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(4267..5082)
                     /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     4990..5009
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5074..5093
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5204..5792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5693..5712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5946..5963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5978..6117
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6043..6062)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6203..6225
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6219..6410)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7219..7308,1..993))
                     /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 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
       61 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
      121 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
      181 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
      241 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
      301 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
      361 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
      421 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
      481 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
      541 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
      601 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
      661 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
      721 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
      781 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
      841 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
      901 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
      961 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     1021 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     1081 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     1141 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     1201 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     1261 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     1321 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     1381 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     1441 gtttaacttt aagaaggaga tataccatgg gcagcagcat taaaatcgcg acccgtaaat
     1501 atctgggcaa acagaatgtc tacgatattg gtgtcgaacg tgaccacaac tttgccctga
     1561 aaaatggctt tatcgcatcc aactgcttta acggtcatca ccaccatcat cacagtacct
     1621 tctccaaact gcgcgaacag ctgggtccgg tgacgcaaga attctgggat aacctggaaa
     1681 aagaaaccga aggcctgcgt caggaaatga gtaaagacct ggaagaagtg aaagccaaag
     1741 ttcaaccgta tctggatgac ttccagaaaa aatggcaaga agaaatggaa ctgtaccgcc
     1801 agaaagtgga accgctgcgt gccgaactgc aggaaggcgc acgccaaaaa ctgcatgaac
     1861 tgcaagaaaa actgtcaccg ctgggtgaag aaatgcgtga tcgcgcccgt gcacatgttg
     1921 acgctctgcg tacccacctg gcgccgtatt cggatgaact gcgtcagcgt ctggccgcac
     1981 gtctggaagc gctgaaagaa aacggcggtg cccgtctggc agaataccat gctaaagcga
     2041 ccgaacacct gtcaacgctg tcggaaaaag ctaaaccggc cctggaagat ctgcgccagg
     2101 gtctgctgcc ggtgctggaa tcatttaaag ttagcttcct gtctgcgctg gaagaatata
     2161 cgaaaaaact gaacacccag ggcacgccgg ttacccagga attttgggac aatctggaaa
     2221 aagaaacgga aggcctgcgc caggaaatgt cgaaagacct ggaagaagtc aaagccaaag
     2281 tgcagccgta cctggacgat tttcagaaaa aatggcaaga agaaatggag ctgtaccgcc
     2341 agaaagtcga accgctgcgc gcggaactgc aggaaggcgc ccgtcagaaa ctgcatgaac
     2401 tgcaggaaaa actgagcccg ctgggcgaag aaatgcgtga tcgtgcccgc gcacatgttg
     2461 atgccctgcg cacccacctg gcaccgtatt ctgatgaact gcgccagcgc ctggcagctc
     2521 gtctggaagc cctgaaagaa aatggcggtg cccgcctggc cgaatatcac gccaaagcca
     2581 ccgaacacct gagtacgctg tccgaaaaag ctaaaccggc gctggaagat ctgcgccaag
     2641 gtctgctgcc ggtcctggaa tcatttaaag tgtcattcct gtcggcactg gaagaataca
     2701 cgaaaaaact gaacacgcag ggcaccccgg ttacccagga attctgggac aacctggaaa
     2761 aggagacgga aggcctgcgt caagaaatga gcaaagatct ggaagaagtt aaagcgaaag
     2821 tccagccgta cctggatgac tttcagaaaa aatggcaaga agaaatggag ttataccgcc
     2881 agaaagttga accgctgcgt gcagaactgc aggaaggcgc tcgtcagaaa ctgcacgagt
     2941 tacaagaaaa actgagtccg ctgggagagg aaatgcgcga ccgtgcccgt gcacatgttg
     3001 atgcactgcg tacgcatctg gcaccgtatt ccgacgaact gcgtcaacgc ctggccgccc
     3061 gcctggaagc gttaaaggaa aatggcggtg cccgtctggc cgaatatcac gcgaaagcca
     3121 cggaacatct gagcacgctg tctgaaaaag ccaagcctgc cctggaagat ctgcgccagg
     3181 ggctgctgcc ggttctggaa agctttaaag tcagtttcct gtccgcgctg gaagaatata
     3241 ccaaaaaact gaatacccag ggttgtctgt cctatgaaac ggaaatcctg acggtggaat
     3301 acggtctgct gccgattggt aaaattgtgg aaaaacgcat tgaatgcacc gtgtatagcg
     3361 ttgacaacaa tggcaacatt tacacgcagc cggtcgcgca atggcatgac cgtggcgaac
     3421 aggaagtgtt tgaatattgt ctggaagatg gttctctgat ccgcgccacc aaagatcaca
     3481 aatttatgac ggtggacggt caaatgctgc cgattgatga aatcttcgaa cgtgaactgg
     3541 atctgatgcg cgttgacaac ctgccgaatt gagatccggc tgctaacaaa gcccgaaagg
     3601 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3661 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     3721 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3781 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3841 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3901 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3961 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4021 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4081 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4141 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     4201 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     4261 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     4321 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     4381 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     4441 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     4501 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     4561 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     4621 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     4681 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     4741 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     4801 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4861 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4921 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     4981 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     5041 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     5101 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     5161 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5221 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5281 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5341 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5401 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5461 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5521 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5581 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5641 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5701 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5761 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5821 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5881 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5941 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     6001 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     6061 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     6121 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     6181 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     6241 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     6301 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     6361 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     6421 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     6481 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     6541 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     6601 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     6661 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     6721 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     6781 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     6841 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     6901 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     6961 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     7021 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     7081 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     7141 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     7201 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     7261 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattggg
//
