LOCUS       sequence-402677-        6096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses split-intein fused MSPdH45 for circularization during 
            expression (internal His-Tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6096)
  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 6096)
  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-cMSPdH45
FEATURES             Location/Qualifiers
     source          1..6096
                     /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(884..901)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1037..1059
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1074..1098
                     /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(1098..1117)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1099..1117)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1201..1220)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1426..1503
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1504..2586
                     /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(1523..1542)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3395..3586
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3580..3602)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3688..3827
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3743..3762
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3842..3859)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4013..4601)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4093..4112)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4712..4731)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4723..5538
                     /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             4723..5538
                     /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(4796..4815)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5630..6085)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5767..5788)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5979..5998
                     /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 cggatctcaa ttcggcaggt tgtcaacgcg catcagatcc agttcacgtt
      181 cgaagatttc atcaatcggc agcatttgac cgtccaccgt cataaatttg tgatctttgg
      241 tggcgcggat cagagaacca tcttccagac aatattcaaa cacttcctgt tcgccacggt
      301 catgccattg cgcgaccggc tgcgtgtaaa tgttgccatt gttgtcaacg ctatacacgg
      361 tgcattcaat gcgtttttcc acaattttac caatcggcag cagaccgtat tccaccgtca
      421 ggatttccgt ttcataggac agacaaccct gggtgttcag ttttttggta tactcttcca
      481 gcgcgctcag aaagctcact ttaaagcttt ccagcaccgg cagcaggccc tgacgcagat
      541 cttccagcgc cggtttcgct ttttcgctca gggtgctcag atgttcggtc gctttcgcat
      601 gatattccgc cagacgcgca ccgccgtttt ctttcagcgc ttccagacgg gccgccagac
      661 gctgacgcag ttcatcgcta tacggcgcca gatgggtacg cagcgcatcc acatgcgcac
      721 gcgcacgatc acgcatctct tcgcccagcg gctgcacttt cgctttcacc tcttccagat
      781 ctttgctcat ttcctgacgc aggccttcgg tttctttttc caggttatcc caaaattcct
      841 gggtcaccgg gcccagctgt tcacgcagtt tgctaaaggt gctgtgatga tggtggtgat
      901 gaccgttaaa gcagttggat gcgataaagc catttttcag ggcaaagttg tggtcacgtt
      961 cgacaccaat atcgtagaca ttctgtttgc ccagatattt acgggtcgcg attttaatgc
     1021 tgctgcccat ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg
     1081 ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga
     1141 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     1201 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     1261 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     1321 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     1381 gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg
     1441 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     1501 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     1561 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     1621 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     1681 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     1741 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     1801 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     1861 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     1921 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     1981 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     2041 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     2101 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     2161 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     2221 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     2281 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     2341 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     2401 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     2461 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     2521 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2581 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc
     2641 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     2701 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     2761 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     2821 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     2881 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat
     2941 gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca
     3001 gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac
     3061 ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa
     3121 gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg
     3181 tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg
     3241 gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg
     3301 ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc
     3361 catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct
     3421 taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct
     3481 ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta
     3541 ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     3601 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     3661 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     3721 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc
     3781 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     3841 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3901 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3961 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4021 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4081 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4141 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4201 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4261 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4321 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4381 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4441 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4501 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4561 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4621 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca
     4681 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     4741 gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     4801 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     4861 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     4921 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     4981 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca
     5041 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     5101 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     5161 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     5221 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     5281 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     5341 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     5401 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     5461 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     5521 ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa
     5581 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     5641 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5701 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5761 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     5821 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5881 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5941 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     6001 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     6061 cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
