LOCUS       sequence-419190-        6225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses split-intein fused MSPdH5 for circularization during 
            expression (external His-Tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6225)
  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 6225)
  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-cMSPdH5
FEATURES             Location/Qualifiers
     source          1..6225
                     /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(1109..1129)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1130..1147)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1166..1188
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1203..1227
                     /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(1227..1246)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1228..1246)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1330..1349)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1555..1632
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1633..2715
                     /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(1652..1671)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3524..3715
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3709..3731)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3817..3956
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3872..3891
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3971..3988)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4142..4730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4222..4241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4841..4860)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4852..5667
                     /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             4852..5667
                     /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(4925..4944)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5759..6214)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5896..5917)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6108..6127
                     /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 ctgggtgttc agttttttgg tatactcttc cagcgcgctc agaaagctca
      541 ctttaaagct ttccagcacc ggcagcaggc cctgacgcag atcttccagc gccggtttcg
      601 ctttttcgct cagggtgctc agatgttcgg tcgctttcgc atgatattcc gccagacgcg
      661 caccgccgtt ttctttcagc gcttccagac gggccgccag acgctgacgc agttcatcgc
      721 tatacggcgc cagatgggta cgcagcgcat ccacatgcgc acgcgcacga tcacgcatct
      781 cttcgcccag cggttccact ttctgacgat acagttccat ctcttcctgc cattttttct
      841 gaaagtcatc cagatacggc tgcactttcg ctttcacctc ttccagatct ttgctcattt
      901 cctgacgcag gccttcggtt tctttttcca ggttatccca aaattcctgg gtcaccgggc
      961 ccagctgttc acgcagtttg ctaaaggtgc taccgttaaa gcagttggat gcgataaagc
     1021 catttttcag ggcaaagttg tggtcacgtt cgacaccaat atcgtagaca ttctgtttgc
     1081 ccagatattt acgggtcgcg attttaatgc cctgaaaata caggttttca tgatgatgat
     1141 gatgatggct gctgcccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga
     1201 ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg
     1261 agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     1321 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     1381 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     1441 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     1501 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     1561 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     1621 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     1681 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     1741 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     1801 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     1861 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     1921 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     1981 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     2041 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     2101 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     2161 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     2221 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     2281 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     2341 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     2401 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     2461 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     2521 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     2581 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     2641 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2701 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     2761 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     2821 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     2881 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     2941 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     3001 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     3061 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     3121 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     3181 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     3241 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     3301 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     3361 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     3421 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     3481 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     3541 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     3601 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     3661 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3721 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3781 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     3841 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     3901 caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     3961 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4021 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4081 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4141 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4201 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4261 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4321 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4381 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4441 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4501 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4561 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4621 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4681 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4741 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4801 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     4861 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     4921 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     4981 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     5041 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     5101 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     5161 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     5221 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     5281 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     5341 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     5401 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     5461 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     5521 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     5581 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     5641 catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg
     5701 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     5761 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5821 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5881 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5941 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6001 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6061 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     6121 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     6181 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
