LOCUS       sequence-171442-        5669 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  bacterial expression of transmembrane proteins using N-terminal 
            MISTIC and C-terminal HIS6 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5669)
  AUTHORS   Vollmer B, Schooley A, Sachdev R, Eisenhardt N, Schneider AM, 
            Sieverding C, Madlung J, Gerken U, Macek B, Antonin W
  TITLE     Dimerization and direct membrane interaction of Nup53 contribute to 
            nuclear pore complex assembly.
  JOURNAL   EMBO J. 2012 Oct 17;31(20):4072-84. doi: 10.1038/emboj.2012.256. 
            Epub 2012 Sep 7.
  PUBMED    22960634
REFERENCE   2  (bases 1 to 5669)
  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: 
            "10.1038/emboj.2012.256"; journalName: "EMBO J"; date: "2012-10-17- 
            17"; volume: "31"; issue: "20"; pages: "4072-84"
COMMENT     Sequence Label: pET28a MISTIC
FEATURES             Location/Qualifiers
     source          1..5669
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             952..1143
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1137..1159)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1245..1384
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1300..1319
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1399..1416)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1570..2158)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1650..1669)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2269..2288)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2280..3095
                     /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             2280..3095
                     /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(2353..2372)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3187..3642)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3324..3345)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3536..3555
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3679..3726)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3722..3740
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3793..3810)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3860..3892)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             complement(3896..3913)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     RBS             4271..4293
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4308..4332
                     /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(4332..4351)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4333..4351)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4435..4454)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4660..4737
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(4757..4776)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
       61 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
      121 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
      181 gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
      241 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
      301 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
      361 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
      421 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
      481 gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt
      541 tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc
      601 tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa
      661 agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga
      721 tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc
      781 tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt
      841 tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc
      901 atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa
      961 caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg
     1021 gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac
     1081 cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     1141 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     1201 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     1261 tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac
     1321 tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     1381 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1441 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1501 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1561 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1621 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1681 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1741 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1801 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1861 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1921 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1981 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     2041 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2101 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2161 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2221 gattttggtc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     2281 tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg
     2341 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     2401 atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     2461 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     2521 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     2581 tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     2641 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     2701 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     2761 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     2821 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     2881 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     2941 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     3001 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     3061 tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata
     3121 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3181 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     3241 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     3301 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     3361 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     3421 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     3481 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     3541 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     3601 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccaatccgga
     3661 tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta
     3721 tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc
     3781 agccggatct cagtggtggt ggtggtggtg ctcgagtgcg gccgcaagct tgtcgacgga
     3841 gctcgaattc ggatccgcga cccatttgct gtccaccagt catgctagcc atatggctac
     3901 cgcgcggcac caggccagaa ccagaacctt ctttttcgcc ttcttcagat actgagatgg
     3961 ataaaatctg tttaataatt gtatttaatt tctcatttag cttttcctgg ccgtacatat
     4021 ctcgggcctg cttcattaac tcggctgtat catcatcaag ctgaataagc ggttcatcaa
     4081 tttccgattc attatacagc tggataaacg cgtccagtcc ttcattcatt cggtcgattg
     4141 ctgtgctcag ctgttccttt tcctcactcg tcactttcat agcttccatc acacccgtgc
     4201 ttttttctaa cagtatgtcc cacttccggt gatgtttttc aaaaaatgta caaaacatgg
     4261 ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     4321 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct
     4381 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
     4441 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
     4501 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
     4561 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     4621 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
     4681 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     4741 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     4801 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     4861 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     4921 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     4981 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     5041 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     5101 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     5161 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     5221 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     5281 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     5341 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     5401 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     5461 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     5521 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     5581 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     5641 gaccgcttgc tgcaactctc tcagggcca
//
