LOCUS       sequence-404922-        6291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses split-intein fused MSP1D1 for circularization during 
            expression (external His-Tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6291)
  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 6291)
  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-cMSP1D1
FEATURES             Location/Qualifiers
     source          1..6291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1291..1482
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1476..1498)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1584..1723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1639..1658
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1738..1755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1909..2497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1989..2008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2608..2627)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2619..3434
                     /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             2619..3434
                     /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(2692..2711)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3526..3981)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3663..3684)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3875..3894
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4018..4065)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4061..4079
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4132..4149)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5167..5187)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5188..5205)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5224..5246
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5261..5285
                     /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(5285..5304)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5286..5304)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5388..5407)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5613..5690
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5691..6291,1..482)
                     /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     complement(5710..5729)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
      541 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
      601 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
      661 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
      721 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
      781 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
      841 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
      901 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
      961 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     1021 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     1081 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     1141 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     1201 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     1261 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     1321 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     1381 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     1441 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     1501 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     1561 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     1621 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     1681 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1741 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1801 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1861 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1921 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1981 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2041 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2101 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2161 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2221 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2281 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2341 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2401 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2461 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2521 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     2581 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     2641 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2701 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     2761 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     2821 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     2881 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     2941 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     3001 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     3061 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     3121 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     3181 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3241 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3301 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     3361 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     3421 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3481 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     3541 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3601 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3661 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3721 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3781 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3841 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3901 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3961 atgcgccgct acagggcgcg tcccattcgc caatccggat atagttcctc ctttcagcaa
     4021 aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt gctcagcggt
     4081 ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatctc agtggtggtg
     4141 gtggtggtgc tcgagtgcgg ccgcaagctt gtcattcggc aggttgtcaa cgcgcatcag
     4201 atccagttca cgttcgaaga tttcatcaat cggcagcatt tgaccgtcca ccgtcataaa
     4261 tttgtgatct ttggtggcgc ggatcagaga accatcttcc agacaatatt caaacacttc
     4321 ctgttcgcca cggtcatgcc attgcgcgac cggctgcgtg taaatgttgc cattgttgtc
     4381 aacgctatac acggtgcatt caatgcgttt ttccacaatt ttaccaatcg gcagcagacc
     4441 gtattccacc gtcaggattt ccgtttcata ggacagacaa ccctgggtgt tcagtttttt
     4501 ggtatactct tccagcgcgc tcagaaagct cactttaaag ctttccagca ccggcagcag
     4561 gccctgacgc agatcttcca gcgccggttt cgctttttcg ctcagggtgc tcagatgttc
     4621 ggtcgctttc gcatgatatt ccgccagacg cgcaccgccg ttttctttca gcgcttccag
     4681 acgggccgcc agacgctgac gcagttcatc gctatacggc gccagatggg tacgcagcgc
     4741 atccacatgc gcacgcgcac gatcacgcat ctcttcgccc agcgggctca gtttttcctg
     4801 cagttcatgc agtttctgac gcgcgccttc ctgcagttcc gcacgcagcg gttccacttt
     4861 ctgacgatac agttccatct cttcctgcca ttttttctga aagtcatcca gatacggctg
     4921 cactttcgct ttcacctctt ccagatcttt gctcatttcc tgacgcaggc cttcggtttc
     4981 tttttccagg ttatcccaaa attcctgggt caccgggccc agctgttcac gcagtttgct
     5041 aaaggtgcta ccgttaaagc agttggatgc gataaagcca tttttcaggg caaagttgtg
     5101 gtcacgttcg acaccaatat cgtagacatt ctgtttgccc agatatttac gggtcgcgat
     5161 tttaatgccc tgaaaataca ggttttcatg atgatgatga tgatggctgc tgcccatggt
     5221 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca
     5281 attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg
     5341 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     5401 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     5461 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     5521 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     5581 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     5641 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     5701 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     5761 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     5821 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     5881 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     5941 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     6001 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     6061 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     6121 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     6181 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     6241 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc t
//
